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A healthy immune system functions through innate and adaptive immunity, the ability to defend against antigens, discriminate between self and nonself, remember previous infections, and cease to attack once the pathogen is removed from the system. The various types of immune cells, their intricate architecture and elaborate regulation, enable the body to maintain homeostasis and activate an adaptive immune response as needed. With flow cytometry, you are able to precisely and rapidly analyze a large number of immune cells, enumerate and characterize them, identify specific surface and intracellular markers of immune subtypes, and assess depletion of immune cells after procedures.
Innate immunity is the primary and immediate cellular response of the body to pathogenic attack through the recognition of conserved features of the pathogens. The recognition of molecular patterns associated with microbes is mediated by pattern-recognition receptors (PRRs), which include both surface receptors (Toll-like receptors (TLRs) and c-type lectin receptors (CLRs)) or cytoplasmic receptors (TLRs, nucleotide-binding oligomerization domain (Nod)-leucine-rich repeat-containing receptors (NLRs), RIG-I-like receptors (RLRs). PRRs recognize two types of ligands—conserved structures of microbial pathogens, known as PAMPs (pathogen associated molecular patterns), and danger signals from altered-self, known as DAMPs (danger associated molecular patterns). Activation of PRRs by their ligands gives rise to the mobilization of innate immune cells and the secretion of signaling and effector molecules (cytokines and antimicrobial peptides) leading to an inflammatory response to neutralize the pathogen or the danger.1 This first line of response is often a prerequisite to trigger initial responses from the adaptive immunity.
Adaptive immunity is elicited through IgGs (B cell receptors) on B lymphocytes (humoral) and T cell receptors (TCRs) on T lymphocytes (cell mediated). Somatic recombination through assembly of variable (V), diversity (D) and joining (J) gene segments enables amazing diversity of antigen recognition by Ig heavy chain and TCR β-chain genes.
Innate immune system comprises primarily of monocytes, macrophages, granulocytes, natural killer (NK) cells and dendritic cells (DCs).
Monocytes are the largest immune cells in the blood. They patrol the body and provide the first line of defense against bacterial and fungal intruders, launch inflammation via cytokine secretion and trigger adaptive immunity. Types of monocytes include classical or inflammatory monocytes characterized by CD14+ CD16− (humans), CCR2+ Ly6Chi (mice) and patrolling monocytes expressing CD14lo CD16+ (humans), CCR2lo Ly6Clo (mice).2
A hallmark of macrophages is their phagocytic capabilities supporting the clearance of intruders and priming immune responses. However, macrophages also enable other essential homeostatic functions such as metabolism.3 Macrophages are highly involved in tumor development and progression as tumor associated macrophages (TAMs) secrete factors inducing immune tolerance towards tumor cells. They are therefore of great interest in immuno-oncology and are central to investigations evaluating the impact of therapy on the tumor microenvironment (TME). Two main types of macrophages are described based on their stimuli induced polarization—M1 and M2. M1 macrophages are pro-inflammatory, promote an oxidative state with reactive oxygen species (ROS) production, and secrete inflammatory cytokines and chemokines, e.g., IL-1, IL-6, IL-12, TNF-α, CXCL9, CXCL10. M2 macrophages are considered immunosuppressive and secrete anti-inflammatory factors such as IL-10 and TGF-β.4
Granulocytes are cells rich in cytoplasmic granules and include basophils5, neutrophils6,7, eosinophils8 and mast cells.9 Granulocytic cells support an array of protective functions including phagocytosis (neutrophils) defense against parasites (basophils) and as effectors in allergy (mast cells) and modulate host responses during immunotherapy.
Natural killer (NK) cells are generally categorized as innate immune cells, however they are similar to lymphocytes in their morphology and in the expression of lymphoid markers. Characteristic markers of NK cells include CD16, CD56 in human and NK1.1 in mice.10, 11
Dendritic cells (DCs), as professional antigen presenting cells, link innate and adaptive immunity by processing and presenting antigens to adaptive immune cells T and B lymphocytes. Three main types of dendritic cells are described:
Conventional markers of dendritic cells include CD11c, BDCA-1/2, CD123.12, 13
Trained immunity or innate immune memory is the property of innate immune cells to adapt the host responses to mount resistance to past intruders.14 The response is still less specific than adaptive immune memory but can allow broad recognition of classes of pathogens such as Gram-positive and Gram-negative bacteria through the involvement of different types of pattern recognition receptors (PRRs).
The B and T lymphocytes are the main components of the adaptive immune arm and launch a delayed but more specific immune response to pathogens.15,16
T lymphocytes or T cells originate in the bone marrow and travel to the thymus for their maturation. Characteristic markers of T cells include CD3 and T cell receptor (TCR). They are later categorized by the expression of other surface molecules CD4 (CD4+ T cells) and CD8 (CD8+ T cells). Flow cytometry screening of surface and intracellular markers allows the distinction of the phenotype and functionality of subsets of T cells: Th1 (CD3, CD4, IFN-g, CCR5, CXCR3); Th2 (CD3, CD4, CCR3, CCR4, CXCR4, IL-4, IL-10); Th17 (CD3, CD4, CCR4, CCR6, TGFbRII, IL-17, CCL20); CD8+ cytotoxic T cells, T-reg or regulatory T cells (CD3, CD4, FoxP3, CD25, CTLA4, OX40/CD134, TGFb, IL-10, IL-35).17
B lymphocytes or B cells start their maturation in the bone marrow. B cells are known for their ability to support humoral immunity through the production of antibodies, but they carry other key functions such as phagocytosis and cross-presentation. As with other lymphocytes, markers of B cells evolve during their maturation and differentiation and include CD19, CD27 BCM, and CXCR4.18
As innate and adaptive immune responses are mounted, they receive support from other body systems and processes including the complement system. The complement system is a network of membrane-bound and soluble protein factors that are engaged in three main pathways: classical complement pathway involving C-reactive protein (CRP), lectin pathway involving collections and mannose-binding lectin (MBL), and the alternative complement pathway. Activation of the complement cascade ultimately leads to cell lysis.19
Flow cytometry is the key technique widely used to identify immune cell populations of interest based on types of surface markers and antigens. In addition to identifying lymphocyte subsets, the technique also enables isolation of rare cell types, detection of intracellular cytokines and quantitative analysis of cells. BD Biosciences offers several research cell sorters, cell analyzers and a comprehensive dye portfolio to support immunology research.
