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Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunology-based therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.

Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunology-based therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.

Immune checkpoint molecules are important regulators of immune activation. This regulation is critical to maintaining immune homeostasis, and is driven by a complex interplay of both co-stimulatory and inhibitory signals. These checkpoints are key to preventing autoimmunity in healthy individuals; however, cancer cells often exploit this system by modulating the expression of these molecules in order to suppress anti-tumor responses during host immuno-editing. The ability to accurately quantify expression levels of checkpoint molecules is critical to ongoing biomedical research efforts exploring potential therapeutic interventions to overcome tumor-mediated immune-evasion. To further aid these ongoing research efforts, we have developed a new bead-based multiplex assay panel specifically designed to quantify several human oncology-related immune checkpoint biomarkers including sCD25 (IL-2Ra), GITR, 4-1BB, sCD27, B7.2 (CD86), free active TGF-β1, CTLA-4, PD-L1, PD-L2, PD-1, TIM-3, LAG-3, Galectin-9. The panel utilizes fluorescence-encoded microsphere technology and is designed for use on commonly available flow cytometers. The panel has been extensively validated for specificity, dilution linearity, cross-reactivity, and inter- and intra-assay precision. This multiplex panel is a robust tool for measuring the concentrations of several immune checkpoint mediators in human serum, plasma, and cell culture supernatant samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA-based quantification methods. Citation Format: Jason Lehmann, Priyanka Rughwani, Amy Zhao, Weiping Jiang, Shaoquan Ji, Binggang Sun. Novel bead-based multiplex assay for the quantification of human immune checkpoint biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4117.

Exposure to foreign material triggers host immune responses, which are primarily mediated by leukocytes including macrophages, neutrophils, dendritic cells, NK cells, T cells and B cells. These cells produce an array of soluble factors which coordinate host defense, tissue repair, and immunoregulatory functions. In addition, these cells and the factors they produce are also critically involved in various diseases including infection, autoimmune disorders, asthma, diabetes, and cancer. Therefore, quantification of these molecules in a cell type-specific context will help achieve in-depth understanding of the immune responses and disease processes. We have developed multiplex assay panels targeting specific mouse immune cells (CD4+ T helper cells, Macrophages/Microglia and B cells), using fluorescence–encoded beads that are suitable for use on common lab flow cytometers. Each panel allows for simultaneously quantification of 13 related analytes as shown in the table below. Cell TypePanel TargetsT helper cellsIL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, TNF-αMacrophages/MicrogliaIL-1β, 6, 10, 12p40, 12p70, 18, 23, TNF-α, CCL22/MDC, CCL17/TARC, CXCL1/KC, G-CSF, TGF-β1B cellsIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, BAFF, BCMA, sCD40 ligand, TGF-β1 Each antibody pair was carefully optimized for assay specificity, sensitivity, accuracy and reproducibility. In addition, these panels have been validated by detecting expected expression level changes of analytes in biological samples cultured under various stimulation conditions. These high quality, low cost and easy to use panels will be useful tools to the study of specific cells involved in innate and adaptive immune responses and the relevant diseases.

Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., T and B cells). Macrophages secrete an array of inflammatory mediators to affect their host defense, tissue repair, and immunoregulatory functions. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-18, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC, MDC, IL-1RA and TGF-β1. We have developed new assay panels targeting human or mouse macrophage/microglia cells, using fluorescence–encoded beads that are suitable for use on commonly available flow cytometers. Each panel allows for the simultaneous quantification of 13 analytes related to macrophage function in humans and mice. These panels have been validated using biological samples to quantify analyte concentration changes in response to various stimuli. These high quality, low cost, easy-to-use panels provide an alternative multiplex solution to the biomedical research community.

Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., B cells). Macrophages secrete an array of cytokines to effect their functions in host defense, tissue repair and immunoregulation. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC and IL-1RA. B cells, in addition to antibody production, also secrete an array of cytokines that mediate Th1- and Th2-like immune responses. These cytokines are produced by regulatory B cells (e.g., IL-10, TGF-β1) and B effector cells, namely Be1 (e.g., TNF-α, TNF-β, IFN-γ, and IL-12p70) and Be2 cells (e.g., IL-2, IL-4, IL-6, TNF-α, and IL-13). Other cytokines associated with activation and survival of B cells such as APRIL, BAFF, and CD40L are also important targets in B cell related processes. We have developed new assay panels targeting macrophage/microglia and B cells, using fluorescence–encoded beads that are suitable for use on general lab flow cytometers. Each panel allows simultaneous quantification of 13 related analytes as shown below. PanelTargetMacrophage/MicrogliaIL-1β, 1RA, 4, 6, 10, 12p40, 12p70, 23, IFN-γ, TARC, TNF-α, Arginase, IP-10B cellIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, TNF-β, BAFF, APRIL, CD40L These panels have been validated by detecting expected changes in biological samples. These high quality, low cost and ease of use panels provide an alternative multiplex solution to the biomedical research community.