Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56 dim NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients' fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients' growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56 dim subset in patients was associated with a lower rate of NK CD56 bright cell proliferation, and the maturation of NK CD56 bright cells toward an NK CD56 dim phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.

Background: Orc1 is the largest subunit of the origin recognition complex that promotes genome duplication. Results: We studied the dynamics of Orc1 during the cell division cycle. Conclusion: Orc1 binds to mitotic chromosomes, and during G 1 phase in the daughter cells it then forms spatial-temporal patterns in the nucleus. Significance: The large subunit of ORC orchestrates the earliest stages of chromosome inheritance.

Cancer is associated with concomitant myeloid cell responses that are characterized by an expansion of tumor associated myeloid cells including, myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs) and neutrophils (TANs). These myeloid derived cells are known to modulate immune responses that enhance cancer cell stemness, angiogenesis, metastasis, invasion and immune escape. The prevailing paradigm is that cancer interferes with hematopoiesis and skews the host system to generate cells of myeloid origin with tumor-promoting functions. Intriguingly, tumor derived factors can perturbate normal bone marrow hematopoiesis and promote extramedullary hematopoiesis in organs such as the spleen. Advances in single cell multiomic technologies now enable the analysis of high dimensional protein and mRNA expression from thousands of cells simultaneously. Using the power of the BD Rhapsody™ Single-Cell Analysis system, we have carefully investigated melanoma associated extramedullary hematopoiesis in mice. To do this, we isolated four hematopoietic stem and progenitor cell populations simultaneously, including Lin- Sca-1+ c-Kit+ (LSK), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocyte-erythrocyte progenitor (MEP) cells from the spleen and bone marrow of melanoma-bearing mice using the BD FACSMelody™ cell sorter in combination with a multiplex BD® AbSeq Oligos panel for surface protein expression and a single-cell whole transcriptome analysis. Using advanced analysis plugins in SeqGeq™ software, we were able to delineate the developmental trajectories of these hematopoietic stem and progenitor cells in this systemic immuno-suppressive myeloid environment. These advanced technologies are critical to uncover tumor mediated abnormalities in hematopoiesis. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Nihan Kara, Xiaoshan Shi, Nikolay Samusik, Stephanie Widmann, Aaron J. Tyznik. A single-cell multiomics approach to study tumor-driven perturbations during hematopoiesis in mice [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 4497.

Cancer is associated with concomitant myeloid cell responses that are characterized by an expansion of tumor associated myeloid cells, including myeloid derived suppressor cells, tumor associated macrophages and neutrophils. The prevailing paradigm is that cancer interferes with normal hematopoiesis by skewing the generation of myeloid cells with tumor promoting functions and promoting extramedullary hematopoiesis in organs such as the spleen. Advances in single cell multiomic technologies now enable the analysis of high dimensional protein and mRNA expression from thousands of cells simultaneously. Using the power of the BD Rhapsody™ Single-Cell Analysis System, we have carefully investigated melanoma associated extramedullary hematopoiesis in mice. To do this, we isolated 4 hematopoietic stem and progenitor cell populations simultaneously, including Lin− Sca-1+ c-Kit+, common myeloid progenitor, granulocyte-macrophage progenitor, and megakaryocyte-erythrocyte progenitor cells from the spleen and bone marrow of melanoma bearing mice using the BD FACSMelody™ cell sorter in combination with a multiplex BD® AbSeq Oligos panel for surface protein expression and a single-cell whole transcriptome analysis. Using advanced analysis plugins in SeqGeq™ software, we were able to delineate the developmental trajectories of these hematopoietic stem and progenitor cells in this systemic immuno-suppressive myeloid environment. These advanced technologies are critical to uncover tumor mediated abnormalities in hematopoiesis. Disclaimers: For Research Use Only. Class 1 Laser Product. BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Summary Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, we describe a simplified intracellular staining workflow for classification of circulating human T and NK cells with cytolytic potential. We suggest reagents for measuring cytolytic proteins and identification of cell subsets within conventional and unconventional T cells and NK cells.

Advancements in cell analysis capabilities have significantly expanded our understanding of the complexity of immunological systems. As the need for deeper analysis has increased, many laboratories have employed a set of lineage markers to define a cell population, followed by the addition of unique markers specific to their biological question. Utilizing regulatory T cells (Treg) as a model population, we describe the design of an 8-color backbone panel on a 12-Color BD FACSLyric™ flow cytometer that can be supplemented with 4 drop-in markers (colors) to characterize two critical facets of Treg biology: activation and homing. Intelligent panel design enabled identification of human naïve and activated Treg cells utilizing established Treg markers (CD3, CD4, CD25, CD127, FoxP3, CD45RA). CD15s and CD161 were included in the 8-color backbone panel to identify functionally suppressive effector and/or pro-inflammatory cytokine secreting Tregs. Importantly, addition of a 4-color drop-in activation panel (PI16, CD147, CD39 and HLA-DR) or a 4-color drop-in homing panel (CCR4, CCR6, CXCR3 and CD31) did not impact resolution of the backbone Treg population(s), while providing new and interesting insights into Treg biology. We highlight the utility of a modular panel design approach that provides an efficient, scalable, and standardized solution for complex analysis of essentially any cell population of interest.

Growing evidence suggests that tumor progression can interfere with normal hematopoiesis and skew the host system to undergo myeloid biased changes. Hematopoietic stem and progenitor cells (HSPCs) are a rare population of precursor cells giving rise to progeny that replenish blood cells throughout life. HSPCs are mainly localized in the bone marrow niche in healthy adults, however pathophysiological conditions such as cancer can cause extramedullary hematopoiesis in organs like the spleen. In this study, we investigated tumor-driven phenotypic and molecular alterations in the HSPC compartment at a single-cell level during extramedullary hematopoiesis using single-cell multiomics. We used a B16-F10 melanoma mouse model to uncover the tumor-mediated changes. Mice with tumor-burden showed splenomegaly and alterations in the composition of the HSPC compartment in the spleen consistent with extramedullary hematopoiesis. We then isolated four HSPC populations, including Lin- Sca1+ cKit+ (LSK), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocyte-erythrocyte progenitor (MEP) cells from the spleen and bone marrow of melanoma-burdened mice using the BD FACSMelody™ Cell Sorter. Next, we used an integrated workflow on the BD Rhapsody™ Single-Cell Analysis System for the downstream single-cell multiomics analysis. We utilized oligo-conjugated antibodies for NGS-based sample multiplexing and protein detection (BD® Single-Cell Multiplexing Kit, BD® AbSeq Assay), along with whole transcriptome analysis for a comprehensive analysis of mRNA expression. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization enabled delineation of the developmental trajectories of HSPC populations and exploration of phenotypic and transcriptomic changes across different hematopoietic subpopulations and across distinct anatomic sites. The analysis also identified unique hematopoietic cell clusters present during melanoma-driven extramedullary hematopoiesis in the spleen. Overall, our findings highlight melanoma-driven perturbations in hematopoiesis by utilizing advanced single-cell multiomics technology. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD-23277 (v1.0) 1120 BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved. Citation Format: Nihan Kara, Nikolay Samusik, Xiaoshan Shi, Chip Lomas, Stephanie Widmann, Aaron J. Tyznik. Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3201.

Advancements in flow cytometry have led to a comprehensive characterization of T cells. While many markers can be used for a detailed immunophenotypic or functional analysis, design of a large multicolor flow cytometry panel is still hampered by the selection of optimal fluorochrome combinations. For example, expansion of existing flow cytometry panels with new markers of interest can result is suboptimal resolution and, in some cases, the need to design a new panel, impacting cost and increasing time to insight. To minimize these challenges and to provide increased flexibility, we have developed a human T cell backbone panel strategically designed to be complemented with 5-8 drop-in markers of choice, depending on instrument configuration. The backbone panel contains five T cell markers (CD3, CD4, CD8, CD45RA, CCR7) conventionally used to identify different maturational states of CD4+ and CD8+ T cells (naïve, central memory, effector memory, effector memory RA). We will show how this backbone panel meets four fundamental requirements: i) clear resolution of major T cell subsets; ii) the fluorochromes used in the backbone panel have minimal resolution impact on the detectors allocated for drop-ins; iii) the fluorochromes assigned to drop-ins have minimal impact on the resolution of the backbone panel; iv) the fluorochromes assigned to drop-ins do not impact each other. We will provide examples of different drop-in combinations for the study of T cell and regulatory T cell biology. Performance of the backbone panel on different instruments will be shown to demonstrate assay consistency across platforms. The compatibility of the backbone panel with intracellular cytokine stain and transcription factor analysis will be further demonstrated. This pre-optimized backbone panel represents a unique tool for researchers who need to study different aspects of T cell biology and want to easily increase their assay capabilities, while minimizing panel design challenges and impact to population resolution. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Citation Format: Mirko Corselli, Chad Sisouvanthong, Nihan Kara, Stephanie Widmann. Development of a human T cell backbone flow cytometry panel enabling flexibility in reagent choice while minimizing panel design challenges [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1392.