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Flow Cytometry and Immunospeak

Rosenthal, Kenneth S. PhD*; Wilkinson, Julie G. MS

Infectious Diseases in Clinical Practice: May 2007 - Volume 15 - Issue 3 - p 183-191
doi: 10.1097/01.idc.0000269912.87477.30
Immunology/Microbiology for ID

The inventions of monoclonal antibodies and flow cytometry brought immunology out of the descriptive and into the biochemical and molecular biological era. These inventions also created a vocabulary and grammar for immunology, immunospeak, that can be confusing and a hurdle to understanding for the outsider. As a tourist to the field of immunology, there are important terms, types of cells, molecules, and responses in immunospeak, which, like words and phrases in a tourist guide, can allow communication with immunology and immunologists. This review provides a description of flow cytometry, monoclonal antibodies, and a tourist guide to immunospeak which describes key cytokines, cells, and cellular determinants that determine immune responses.

*Northeastern Ohio Universities College of Medicine, Rootstown, OH and †Beckman Coulter, Inc, Custom BioPharma Solutions, Miami, FL.

The author (K.S.R.) has research support from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

This is the fifth in a continuing series of articles in the new Immunology and Microbiology: Clinical Correlations section. The purpose of this section is to highlight basic science explanations for microbial disease, disease signs, and symptoms, immunology, and interventions. We welcome your input regarding potential topics for discussion, and they can be relayed to my e-mail address,

Address correspondence and reprint requests to Kenneth S. Rosenthal, PhD, Northeastern Ohio Universities College of Medicine, 4209 SR 44, Rootstown, OH 44272. E-mail:

Immunology has baffled many very good microbiologists and even some physicians. Maybe the confusion arises because immunology is a system that is turned on by the trash and debris that is left by microbial invaders and where an active response is critical to the cure but is often the cause of the disease. The same molecules that activate some responses inhibit others, and it is a system that requires tolerance, but immunotolerance is all about suppression. As with many sciences, even microbiology, the vocabulary is a big hurdle toward understanding. For immunology, this is even truer. Immunospeak, as I call it, is a language of CD numbers, cytokines, chemokines, and all "kines" of small molecules; major histocompatibility complex (MHC) compatibilities and incompatibilities; and cascades and circuits of activations and inhibitions. The words of immunospeak are defined now by monoclonal antibodies as determined by the technique of flow cytometry, although, historically, the characterization of immunomolecules was via functional cellular assays (eg, migration studies to define chemokines and their receptors). This article will provide a brief introduction to monoclonal antibodies, flow cytometry, and flow cytometric analyses and then a look at the basic immunodefinitions (identified in bold-faced text) of immunospeak. More description of how an immune response is generated is explained in "Vaccines Make Good Immune Theater: Immunization as Described in a Three-Act Play."1

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Before the invention of monoclonal antibodies by César Milstein, Niels K. Jerne, and Georges Köhler, the different types of white blood cells were distinguished primarily by histological staining, morphology, and biochemical tests. Immunology textbooks were limited to the discussion of the properties and activity of antibodies because cellular responses were too difficult to describe. Cellular immunology was an indecipherable genetic analysis of mouse immune responses defined by the mouse strains that could or could not react with arsenobenzene and like compounds. T lymphocytes could be distinguished from B lymphocytes because T cells would bind to sheep red blood cells (RBCs), forming a halo of RBCs around the T cell was called a rosette. Human cellular immunology could not be discussed because of the lack of experimental subjects.

Monoclonal antibodies changed everything. The good news and the bad news about a monoclonal antibody are that it is very specific. This is good because it can distinguish individual antigens on the cell surface but bad because it is so specific that several different monoclonal antibodies will describe the same molecule, and a slight variation in the molecule can preclude recognition. Monoclonal antibodies are produced by cells that are created in the laboratory by the fusion of an antibody-producing B lymphocyte from an immunized mouse with an immortalized myeloma cell to produce a hybridoma cell. This is followed by the detection, isolation, and growth of the hybridoma of interest-a difficult time encompassing process. Once cloned and grown, the hybridoma cell will make the antibody of interest and live forever (or until the culture gets contaminated with mycoplasma or there is a power failure).

The monoclonal antibodies that distinguish immune cells are directed at cell surface molecules. The first antibodies to be developed for human immunology discerned T cells from B cells. Then, it became apparent that monoclonal antibodies could distinguish subsets of T cells. To add to the confusion of the early years, different people and different manufacturers had different monoclonal antibodies that would distinguish the same cell. Because these antibodies could be recognizing different parts of 1 molecule and possibly different molecules of a complex, the antibodies were then grouped together based upon their antigenic target, and the target was termed a cluster of differentiation, the well-known CD number. Creation of the catalog of CD numbers for these new monoclonal antibodies and their cellular targets was the basis for the first edition of the Immunospeak Dictionary. Ellis Reinherz,2 his colleagues at the Dana Farber Cancer Center, and others initiated the Dictionary of Human Immunospeak by developing some of the earliest monoclonal antibodies to T cells. They characterized antibodies that recognized all T cells, such as antibodies to the sheep RBC rosette receptor, CD2, or to the CD3 complex of proteins. Then, they characterized the antibodies that recognize the individual proteins that distinguish helper and killer T cells to define the CD4 and CD8 T subsets. As of the 8th HLDA Workshop held in 2004, there are more than 350 different CD designations, each of which is defined by a group (or cluster) of monoclonal antibodies. Specific cell types and function can be described by the presence of one or more of these CD molecules, providing what we call in immunospeak an immunophenotype. The process of doing this is called immunophenotyping. Adding to a beginner's confusion is the use of a single CD name to describe the monoclonal antibody specificity, the antigen detected, and the cell type. For example, the CD3 complex of proteins on the surface of T cells (CD3 cells) can be detected by the CD3 (not anti-CD3) monoclonal antibody. Cytokines and chemokines have tried to avoid this confusion through the use of slightly different names for the cytokine molecule (eg, interleukin [IL] 7) and the cell surface cytokine receptor protein (eg, IL-7R for receptor) (see below). Unfortunately, immunologists speak often in a shorthand version of immunospeak and will refer to a cell with the IL-7R on the surface as simply an IL-7-positive cell. The devil is in the details.

At about the same time that monoclonal antibodies were being developed, the flow cytometer (FCM) and its bigger cousin, the fluorescence-activated cell sorter (FACS) entered the field of immunology. When I (K.S.R.) was a postdoc in 1977 to 1979, there were only 7 commercial FACS units in the United States, and 2 were at the Harvard Medical Complex. I snuck in some time on the unit at the Dana Farber Cancer Center until the director of the department objected and then briefly used the unit in the Pathology department until I had the privilege of working with Howard Shapiro, MD. Howard is one of the initial developers of flow cytometry and is responsible for enhancing the use of flow cytometry and making it one of the most scintillating of all sciences. His book is the definitive guide to flow cytometry,3 and a well-thumbed copy is next to every FCM.

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An FCM is basically an automated fluorescent microscope that can analyze size, granularity, and several different fluorescent parameters of an individual cell as captured in time (Fig. 1). Unlike the microscope, the light source is a laser, the cells are illuminated by the laser beam as they flow within a high-pressured sheath fluid instead of being on a slide, and the light emitted or scattered by the cells is digitally analyzed by photomultipliers and computers instead of by the eye. The cells are focused into a single-file progression in front of the laser by injection into the high-pressure stream of sheath fluid. The laser light hits each cell and either is scattered or excites the fluorescent molecules on or within the cell. The scattered or fluorescent-emitted light is then guided by mirrors and filters to different photomultiplier tubes for analysis. Many commercial units use more than 1 laser and can evaluate 7 or more parameters for each cell (forward light and side light scatter, time, and 4 or more colors).



Light scattering of the coherent light of the laser is used to discern the size and granularity of cells. The amount of light scattered at a small angle (forward scatter [FS]; eg, 5 degrees) is proportional to the size of the cell. Laser light may also bounce off of vesicular/granular objects within cells and be scattered in all directions, including perpendicular to the incident beam (side scatter [SS]). This allows evaluation of the granularity of cells. The combination of forward light scatter and side light scatter analyses can be used to provide a differential white blood cell analysis (Fig. 1).

The light emitted by fluorescent probes is usually analyzed at right angles to the laser beam. Fluorescent molecules are excited by a range of light wavelengths and emit light at longer wavelengths. The wavelength of light emitted by the laser determines the types of fluorescent probes that can be used. For example, most FCMs use an argon ion laser which emits a 488-nm wavelength beam capable of exciting molecules such as fluorescein and rhodamine, molecules that are commonly attached to antibodies for immunophenotyping, and propidium iodide, which is used to quantitate intracellular DNA. The classic fluorescein label that is attached to antibodies for immunofluorescence is excited by blue light and emits green light. Helium cadmium lasers produce UV light and can excite DAPI (4′,6-diamino-2-phenylindole) which binds to DNA, whereas helium neon red lasers are used to excite red-emitting fluorochromes that can also be attached to antibody molecules. The artistry of a flow cytometry technician and the sophistication of the instrument are demonstrated by the number of different fluorescent colors that can be analyzed simultaneously because the emission spectra of the fluorochromes overlap, and the overlap requires electronic/computer compensation to ensure that data are not being created or lost by the instrument.

The immunophenotype of a cell can be determined using antibodies that are covalently labeled with a variety of fluorescent molecules (fluorochromes; Fig. 2). There has been an explosion in recent years of the quantity and quality of fluorescent compounds that can be used for flow cytometry. The traditional fluorochromes that are attached to antibodies were fluorescein and rhodamine, but these have been supplemented with Texas Red, phycoerythrin, Cyanines (eg, Cy-5), Pacifics, Alexas, and combinations of fluorochromes (tandems [eg, phycoerythrin + Cy5]). Newly developed fluorescent nanoparticles4 (trademarked Q-dots) may replace fluorochromes for flow cytometry. Q-dots are made of a core semiconductor nanoparticle containing a fluorochrome which is encapsulated in a shell that allows covalent attachment of antibodies and other proteins. The nanoparticles have many spectroscopic advantages for flow cytometry.



Cells can be analyzed for many different parameters besides their immunophenotype including DNA content, DNA/RNA/protein content, enzyme function, signal transduction, or even membrane potential, depending on the type of fluorescent probe that is used. Evaluation of individual cellular DNA content provides a description of the cell cycle for a population of cells. A fluorochrome, like propidium iodide, that binds specifically to DNA in a stoichiometric manner can distinguish cells in G0/G1 phase that have 2 sets of chromosomes from cells in G2 phase that have 4 sets of DNA and from cells in S, which have intermediate amounts of DNA (Fig. 3A). Normal cells, including peripheral blood mononuclear cells (PBMCs) and other cells, are not proliferating and will only have 2n DNA. Cancer cells are growing and often have altered chromosomes such that the cell cycle measurement will show intermediate amounts or greater than 4N DNA per cell indicative of aneuploidy. Cell cycle measurement can be prognostic of certain cancers (breast cancer ploidy and S-phase).5,6 Further analysis of the cell cycle can be provided by quantitating RNA or protein with appropriate fluorochromes or with antibody to specific markers of the stages of the cell cycle other than the DNA content (Fig. 3B).7 Membrane potential can be depicted by the uptake or release of charged fluorescent molecules as the membrane potential changes. Similarly, phagocytosis or enzyme activity can be demonstrated by the accumulation or conversion of a molecule into a fluorochrome that becomes trapped in the cell. The possibilities of flow cytometry are limited only by your imagination.



The FACS is a fancy FCM that can separate cells based on the parameters defined by the user and identified by the FCM. When the FCM determines that a cell with the selected parameters has passed through the laser beam, it will put an electric charge on the sheath fluid in the flow cell and then ultrasonically break the stream into defined droplets large enough to carry a cell. The stream passes through a set of positively and negatively charged electrostatic plates that will deflect or pull the droplet aside into a collection vessel. This method can sort cells at an amazing rate of 50,000 to 100,000 cells/s, but cells are traditionally sorted at a speed of 5000 cells/s. Although this traditional rate seems high, if the desired cell population were 1% of the total, it would still take approximately 5 hours to collect 106 cells.

The flow cytometric data is usually presented as a histogram for 1 parameter (Fig. 3) or a dot blot scattergram for 2 parameters (Fig. 2). Basically, the histogram is a plot of the number of cells on the y axis that have a specific quantity of signal. This may be the amount of light scattering, DNA-related fluorescence or immunofluorescence, on the x axis. Each dot on the dot blot represents a cell or a multiple of cells that share both parameters.

As can be seen in Figure 2, PBMC with very different light-scattering properties can be distinguished easily by flow cytometry. In addition to quantitating these cells, the FCM's computer allows us to pick a population of these cells for further analysis that is akin to an electronic separation of cell types. This population of cells will be identified by circling the region of interest on a dot blot or the histogram corresponding to the parameters of the cells of interest. Flow cytometrists call this setting gates or gating. Once identified in this manner, the number of cells within the "gates" (gated region) can be obtained, and the computer can be told to focus on only these cells and provide a further description of the other parameters it has collected from these cells. For example, as seen in Figure 2, cells identified as lymphocytes by their size and lack of granularity can be distinguished further as CD4 T-helper cells or CD8 T-cytotoxic cells using monoclonal antibodies. Alternatively, the CD4 T cells can be analyzed for other parameters including cell activation by the presence of CD69 or CD25 (the alpha subunit of the IL-2 receptor) on the surface of cells. The CD4 T cells can also be further subtyped as regular CD4 T cells or T regulatory cells based on the high expression of CD25 and low expression of CD127 (IL-7 receptor alpha chain)8 in the absence of obvious activators.

In addition to the cell surface markers that identify the different immunologic cells, cells can also be permeabilized to allow analysis of intracellular and intranuclear molecules. For example, activated CD4 T cells could be evaluated for production of the cytokine, interferon (IFN) γ. To make it possible to detect the cytokine produced by an activated lymphocyte, the cells would be incubated with an inhibitor of vesicular transport (Brefeldin-A), so that the cells would concentrate and not release the cytokine. The cells would be treated with monoclonal antibodies to CD4 and CD69 that are labeled with different colored fluorescent molecules, lightly fixed with formaldehyde and then permeabilized with a weak detergent, such as saponin, before the addition of an antibody to IFNγ, labeled with a third fluorochrome. Representative data are shown in Figure 2.

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The first lesson in immunospeak is that almost all molecules can elicit an immune response. Immunologists described the components of the immune system with antibodies even before they were isolated or defined as proteins or carbohydrates. For example, MHC proteins are often called MHC antigens (see below). Even immunoglobulin (Ig; the antibody molecule) will elicit immune responses to produce characteristic antibodies to different parts of the molecule. Before the structures of the immunoglobulins were known, their antigenic differences were described by antibodies, and this created probably one of the most confusing parts of immunospeak. The different types (isotypes) of immunoglobulins, IgM, IgD, IgG, IgE, and IgA, have very different protein structures, and immunization of a mouse with purified human IgM will produce antibodies that will distinguish human IgM from IgG, IgE, and IgA. Similarly, there are 4 different structures for IgG and 2 for IgA (subtypes). Within each immunoglobulin molecule are structural regions that vary from person to person because "all 'o us are different," and these will elicit antibodies that distinguish allotypes. Finally, each of the different types of immunoglobulin (eg, IgG) is a collection of molecules with a protein region that recognizes different antigenic structures (epitopes). These immunoglobulin protein regions can also be defined by an antibody. There are many of these idiotypes (just as there are many "idiots" in the world).

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The cell surface proteins, including the MHC (also known as HLA in humans) molecules, on immune cells are the costumes worn by the cells that allow us to identify and distinguish different cells. As a costume, these molecules also tell us about the role/function of the cells.

The MHC is actually a chromosomal locus that contains the genes for several immunologic proteins. The MHC region was discovered in the early immunogenetic studies of tissue graft acceptance and rejection. The MHC molecules include the histocompatibility antigens that must be matched for a successful tissue transplant. Within this genetic region are encoded the MHC I and II proteins (usually referred to as antigens). The biochemical characterization and radiograph crystallographic description of MHC I and II molecules by Jack Strominger and Don Wiley's laboratories showed that the MHC molecules bind small peptides degraded from larger antigenic proteins and display these peptides to T lymphocytes. Each of our cells express MHC proteins obtained equally from each parent. The MHC class II proteins are encoded within 3 regions called DP, DQ, and DR and are expressed primarily on macrophages, B cells, and dendritic cells. These cells are grouped as antigen-presenting cells (APCs) because they present foreign and other proteins to CD4 T cells. The human MHC class I molecules include the classic HLA A, HLA B, and HLA C molecules and nonclassic HLA molecules. The MHC I molecules are present on all nucleated cells and display peptides indicative of the protein repertoire of the cell to CD8 T cells. As a result, MHC I molecules inform the CD8 T cell of the presence of an intracellular infection with a virus or identify the cell as a tumor or transplanted cell. Cancer and some viruses can be very sneaky and hide the MHC molecules so the immune system is blind to their presence.

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Most cell surface determinants on immune cells are named by immunologists with CD numbers. To an immunologist, a CD is not a recording and not a bank transaction but a namefor a molecule or complex of molecules, as defined by a group of monoclonal antibodies. The CDs take up a large portion of the Immunospeak Dictionary. Many of these CDs define cell populations, which may also define functions for these cells, whereas other CDs fill up the literature with attempts to describe their purpose. There are more than 350 CDs in the dictionary now. Several of the most important CDs, their cell type, and function are listed in Table 1. To add to the confusion, many molecules are known by functional names and CDs. For example, CD127 is still referred to as IL-7 receptor chain alpha, CD71 is the transferrin receptor, and CD235a on RBCs is still called glycophorin-A. The MHC molecules do not require CD designations.



Immunologists have become very dependent upon their ability to describe cell types by an immunophenotype with a dog tag consisting of CDs. The antibodies recognizing these CDs have become the basic tools for identifying and quantitating these cell types in blood or other sample by flow cytometry. For example, CD2 and CD3 are useful markers for T cells, CD19 and CD21 for B cells, and CD56 for natural killer (NK) cells.

Immunologists like to assign function to cell types based on their name and CD dog tag, but Mother Nature complicates the issue. Historically, the textbooks defined the CD4 T cells as helper T cells and the CD8 T cells as killer and suppressor T cells. Simplistically, CD4 helper T cells make cytokines that influence the nature of the immune response, and CD8 cytotoxic T cells kill virus-infected and tumor cells (hence the name "cyto" for cell and "toxic" for death inducing). Unfortunately, these cells did not read the textbook; CD8 T cells can make cytokines, and CD4 T cells have mechanisms for killing target cells. Initially, helper T cells were further divided into TH1 and TH2 subsets based on the types of cytokines that they produce9 (see below). Additional CD4 subsets, including TH3, TH17, and Treg, have since been identified. Except for the Treg cells that express CD4 and CD25 designations, these subsets are not readily discernible by a cell surface marker or CD designation. To add to the confusion, CD8 T cells and dendritic cells are being divided into Tc1 and Tc2, and DC1 and DC2 subsets, respectively, based on subtle differences in function which are defined by a signature (or dog tag) rather than individual markers.

Using CDs to identify specific cell types can be confusing sometimes because the marker may be present in smaller amounts on other cell types or expressed only under special conditions. The MHC II molecule provides a good example of the latter situation. The MHC II is important for antigen presentation and was initially thought to be a B-cell specific marker; then, it was discovered on other APCs such as macrophages and dendritic cells. Only Mother Nature knows why the MHC II molecule is also present on activated T cells.

Some cells are described by the lack of a CD, by bright or dim CD immunofluorescence per FCM, or by other markers that lack CD numbers. Natural Killer cells (NK) were first described by their lack of markers. NK cells were called non-T, non-N cells based on their lack of typical B and T cell markers. Now, NK cells are identified by their expression of CD56 and killer cell immunoglobulin-like receptors molecules. The NK cells share several CDs and functions with T cells and more recently, natural killer T cells (NKT) were discovered that resemble the NK cell more than other T cells but are distinguished from the NK cell by the presence of a T cell receptor (TCR) (an identity crisis even for an immunologist). The TCR on T cells recognizes the antigenic peptide presented on an MHC molecule. The TCR and killer cell immunoglobulin-like receptors do not have CD numbers.

Unfortunately, CDs are not available to define all relevant cell types. For example, good CD markers for distinguishing the different types of dendritic cells are still not available. Dendritic cells (usually referred to as DCs, but I will not use the abbreviation because it looks so much like CDs) were originally defined histologically and morphologically by the presence of tendrils on the cell. Now, dendritic cells are distinguished by their different origins and functions as myeloid, lymphocytic, plasmacytoid, Langerhans, and follicular dendritic cells. Dendritic cells require several dog tags to be identified such as the presence of MHC II, CD11c, CD123, CD85k, CD14, CD80, CD86, or DC-SIGN and the absence of CD3, CD56, and CD19 (as an immunologist it is socially acceptable to be an immunological CD name dropper). These very important cells are the directors of the innate and adaptive immune responses. Dendritic cells produce important cytokines, present antigen to lymphocytes, and, except for follicular dendritic cells, are required for initiating an immune response. Many vaccine producers have been studying dendritic cells as a natural delivery vessel of either an engineered or a natural antigen. Follicular dendritic cells are sticky and bind proteins for display to B lymphocytes but cannot present antigen to T cells because they do not express MHC II molecules. Myeloid dendritic cells are further divided into DC1 and DC2 cells (cannot avoid the abbreviation here) based on how they were activated, the cytokines that they produce, and whether they induce a TH1 or TH2 response in CD4 T cells. DC1 dendritic cells carrying tumor antigens can be used as anti-cancer vaccines to activate anti-tumor immunotherapies.10,11 Clear immunophenotypes characterized by cell surface CD markers for the different types of dendritic cells would simplify this very important and growing area of immunology.

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Another large section of the Immunospeak Dictionary includes cytokine and chemokine proteins. Cells are blind, so these proteins provide molecular communication for the immune system. Cytokines are like hormones that bind to specific receptors to manipulate the function of cells and the immune response. Chemokines are a subset of cytokines and are also attractants for immune cells. These messenger proteins are usually identified by tests that use monoclonal antibodies. Immunologists drop these C words in every conversation, expecting that the rest of us know which cells are the producers, the nature of the stimulus, the target cell that the cytokine binds to, and the outcomes. Table 2 provides this information for selected cytokines for those of us who are not fluent in this component of immunospeak.12



As seen in Table 2, the cytokines can be divided into different groups based on the type of responses that they induce. These include the acute-phase response, TH1, TH2, TH17, Treg, and other responses. The acute-phase response is usually triggered by bacterial infections and promotes fever, sepsis, and other consequences. The TH1 responses activate cellular and antibody components to reinforce local inflammatory responses to infection, especially by intracellular microorganisms; promote delayed-type hypersensitivity (eg, poison ivy type) responses; and promote cell-mediated autoimmunity. The TH17 responses are also triggered by bacterial infections and, through the release of IL-17, support local inflammatory reactions. The TH2 responses activate antibody responses, including IgE-mediated allergic responses. The Treg cells produce suppressive cytokines that prevent T cell and macrophage activation unless overridden by an appropriate set of cytokine and cellular triggers.

As with many languages, the tourist can get by with a limited cytokine vocabulary. For immunospeak, the essential cytokine vocabulary includes IL-1, tumor necrosis factor (TNF) α, IL-12, IFNγ, IL-2, IL-4, IL-10, and transforming growth factor (TGF) β (Table 2).

The IL-1 and TNF are acute-phase cytokines produced by macrophages in response to bacterial infection. These cytokines kick start protective responses and symptoms of sepsis and chronic inflammation including fever, acute-phase responses, increased vascular permeability, increased adhesion molecules on vascular endothelium, fibroblast proliferation, and T- and B-cell activation. The TNF comes in TNFα and TNFβ varieties, and other cells can make these cytokines. The TNFα is also known as cachectin also causes loss of appetite and weight loss. If harnessed, this could be the next miracle drug for Hollywood. The TNF affects many different cell types and is responsible for the detrimental effects of sepsis and promoting the inflammation associated with rheumatoid arthritis (RA). Antibody or soluble receptor antagonists of TNF are available to block the cycle of inflammation caused by TNF. The TNFα is such a potent molecule and causative agent in diseases such as RA, that there are 2 very effective biological therapies directly targeting either the TNF cytokine or the TNF cytokine receptor.

The IL-12 and IFNγ work together to reinforce local antimicrobial inflammatory responses by promoting cellular protections. The IL-12 is produced by monocytes, macrophages, and dendritic cells. It is initially produced in response to the presence of bacteria and some other microbes, and it activates the production of IFNγ by T helper cells (CD4). The IFNγ is the keystone cytokine of a TH1 type of T helper cell response. The TH1 response can be remembered by the following: 1 means first, promoting the early responses necessary to control an infection, and these include antibody and cell-mediated responses. The IFNγ will activate macrophages and promote the production of more IL-12 by the cells listed above to establish a cycle of cell-mediated immune activations. Reinforcement of this cycle is essential for control of viral, mycobacterial, leishmanial, other intracellular infections, and antitumor responses. The IFNγ also provides support for the maturation of antibody production from IgM to IgG. The IL-12 will also activate NK cells that can attack virus-infected cells and support antibacterial responses. When Mother Nature throws a monkey wrench into the delicately balanced immune system, the IL-12/IFNγ cycle will reinforce autoimmune cellular responses, as in RA and multiple sclerosis, and be debilitating to the person.

The IL-2 is a lymphocyte growth factor produced as part of the TH1 type of response. The IL-2 promotes the growth of T, B, and NK cells to expand the immune response. The IL-2 cytokine was one of the first immunotherapies that proved to be a wonder drug but with many deleterious side effects because of the immune response running wild after being stimulated.

The IL-4 is the keynote cytokine associated with promoting TH2 types of helper T-cell responses. The IL-4 is the "default" of TH2 cellular responses and is initiated in the absence of IL-12. In a natural response to infection, the TH2 comes after TH1, second, and later, and it is important for systemic protections. The TH2 responses are primarily antibody and not cell-mediated responses. The IL-4, in combination with IL-5, will promote maturation of antibody production from IgM to IgG, IgE, and IgA. The IL-5 is often called the "allergy" cytokine because of the direct involvement with IgE production. Systemic protections are provided by antibody. Inactivated vaccines usually elicit TH2 type of responses. The TH2 types of responses are also important for defense against worm infections. The IL-4 is a lymphocyte growth factor and reinforces the production of more IL-4 by T helper cells and prevents the production of TH1 cytokines.

The IL-10 and TGFβ are immunosuppressive cytokines. The IL-10 is produced as part of a TH2 response and prevents TH1 responses. The TGFβ is produced by Treg and other cells to suppress T cell and macrophage activation and prevent spurious responses.

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Oftentimes, the grammar complicates learning a new language. For example, German sentence structure adds difficulty to understanding German. Similarly, the grammar of immunospeak can complicate the speaking and understanding of the language. Immunospeak grammar consists of the networks of cellular and molecular activators, regulators, suppressors, and mediators of the immune response. As indicated above, the immune system is an autoregulating system controlled by the binding of cells or cytokines to multiple cell surface receptors and the activation of many different cascades of kinases and phosphatases and calcium ion fluxes. The culmination of these pathways results in the activation or inhibition of nuclear factors (eg, NFκβ) to produce immune molecules that promote cellular growth, induction of apoptotic pathways, or cell differentiation (life, death, and the pursuit of happiness). These pathways can be envisaged as the resultant overlay of subway maps from every major city in the world; therefore, further discussion of these interconnecting cross-regulating pathways is beyond the scope of this review.

The vocabulary and grammatical rules of immunospeak continue to evolve daily as this exciting field of immunology grows. New discoveries add new words, and new grammar and immunologists continue to provide new definitions and rules for these words and grammar.

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The authors thank Enrique Rabellino and Sybil D'Costa for the review of the manuscript. The authors also thank Howard Shapiro for exciting our interest in flow cytometry.

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