Hematopoiesis and Cells of the Immune System
Stem cells are defined by two capacities: (1) the ability to regenerate or “self-renew” and (2) the ability to differentiate into diverse cell types. Embryonic stem cells have the capacity to generate almost every specialized cell type in an organism (in other words, they are pluripotent). Adult stem cells, in contrast, have the capacity to give rise to the diverse cell types that specify a particular tissue (they are multipotent). Multiple adult organs harbor stem cells that can give rise to cells specific for that tissue (tissue-specific stem cells). The HSC was the first tissue-specific stem cell identified and is the source of all of our red blood cells (erythroid cells) and white blood cells (leukocytes).
Hematopoietic Stem Cells Differentiate into All Red and White Blood Cells
HSCs originate in fetal tissues and reside primarily in the bone marrow of adult vertebrates. A small number can be found in the adult spleen and liver. Regardless of where they reside, HSCs are a rare subset—less than one HSC is present per 5 ×104 cells in the bone marrow. Their numbers are strictly controlled by a balance of cell division, death, and differentiation. Their development is tightly regulated by signals they receive in the microenvironments of primary lymphoid organs.
Under conditions when the immune system is not being challenged by a pathogen (steady state or homeostatic conditions), most HSCs are quiescent; only a small number divide, generating daughter cells. Some daughter cells retain the stem-cell characteristics of the mother cell—that is, they remain self-renewing and are able to give rise to all blood cell types. Other daughter cells differentiate into progenitor cells that have limited self-renewal capacity and become progressively more committed to a particular blood cell lineage. As an organism ages, the number of HSCs decreases, demonstrating that there are limits to an HSC’s self-renewal potential.
When there is an increased demand for hematopoiesis, for example, during an infection or after chemotherapy, HSCs display an enormous proliferative capacity. This can be demonstrated in mice whose hematopoietic systems have been completely destroyed by a lethal dose of x-rays (950 rads). Such irradiated mice die within 10 days unless they are infused with normal bone marrow cells from a genetically identical mouse. Although a normal mouse has 3 ×108 bone marrow cells, infusion of fewer than 104 bone marrow cells from a donor is sufficient to completely restore the hematopoietic system. Our ability to identify and purify this tiny subpopulation has improved considerably, and in theory we can rescue the immune systems of irradiated animals with just a few purified stem cells, which give rise to progenitors that proliferate rapidly and repopulate the blood system.
Because of their rarity, investigators initially found it very difficult to identify and isolate HSCs. Classic Experiment Box 2-1 describes experimental approaches that led to the first successful isolation of HSCs. Briefly, these efforts featured clever process-of-elimination strategies. Investigators reasoned that undifferentiated HSCs would not express surface markers specific for mature cells from the multiple blood lineages (“Lin” markers). They used several approaches to eliminate cells in the bone marrow that did express these markers (Lin+ cells) and then examined the remaining (Lin–) population for its potential to continually give rise to all blood cells over the long term. Other investigators took advantage of two technological developments that revolutionized immunological research—monoclonal antibodies and flow cytometry (see Chapter 20)—and identified surface proteins, including CD34, Sca-1, and c-Kit, that were expressed by the rare HSC population and allowed them to be isolated directly.
We now recognize several different types of Lin– Sca-1+c-Kit+ (LSK) HSCs, which vary in their capacity for self-renewal and their ability to give rise to all blood cell populations (pluripotency). Long-term HSCs (LT-HSCs) are the most quiescent and retain pluripotency throughout the life of an organism. These give rise to short-term HSCs (ST-HSCs), which are also predominantly quiescent but divide more frequently and have limited self-renewal capacity. In addition to being a useful marker for identifying HSCs, c-Kit is a receptor for the cytokine SCF, which promotes the development of multipotent progenitors (MPPs); these cells have a much more limited ability to self-renew, but proliferate rapidly and can give rise to both lymphoid and myeloid cell lineages.
HSCs Differentiate into Myeloid and Lymphoid Blood Cell Lineages
An HSC that is induced to differentiate ultimately loses its ability to self-renew as it progresses from being an LT-HSC to an ST-HSC and then an MPP (Figure 2-2). At this stage, a cell makes one of two lineage commitment choices. It can become a myeloid progenitor cell (sometimes referred to as a common myeloid progenitor or CMP), which gives rise to red blood cells, platelets, and myeloid cells (granulocytes, monocytes, macrophages, and some dendritic cell populations). Myeloid cells are members of the innate immune system, and are the first cells to respond to infection or other insults. Alternatively, it can become a lymphoid progenitor cell (sometimes referred to as a common lymphoid progenitor or CLP), which gives rise to B lymphocytes, T lymphocytes, innate lymphoid cells (ILCs), as well as specific dendritic cell populations. B and T lymphocytes are members of the adaptive immune response and generate a refined antigen-specific immune response that also gives rise to immune memory. ILCs have features of both innate and adaptive cells.
FIGURE 2-2 Regulation of hematopoiesis by transcription factors. A large variety of transcription factors regulate hematopoietic stem cell (HSC) activity (quiescence, self-renewal, multipotency) as well as differentiation to the several lineages that emerge from HSCs. Several key factors are shown here. Note that erythrocytes and megakaryocytes may arise not only from myeloid progenitors, but also from the earliest hematopoietic stem cell populations. Hematopoietic regulation is an active area of investigation; this is one possible schematic based on current information.
Recent data suggest that precursors of red blood cells and platelets can arise directly from the earliest LT- and ST-HSC subpopulations (see Figure 2-2). Indeed, the details behind lineage choices are still being worked out by investigators, who continue to identify intermediate cell populations within these broad progenitor categories.
As HSC descendants progress along their chosen lineages, they also progressively lose the capacity to contribute to other cellular lineages. For example, MPPs that are induced to express the receptor Flt-3 lose the ability to become erythrocytes and platelets and are termed lymphoid-primed, multipotent progenitors (LMPPs) (Figure 2-3). As LMPPs become further committed to the lymphoid lineage, levels of the stem-cell antigens c-Kit and Sca-1 fall, and the cells begin to express RAG1/2 and TdT, enzymes involved in the generation of lymphocyte receptors. Expression of RAG1/2 defines the cell as an early lymphoid progenitor (ELP). Some ELPs migrate out of the bone marrow to seed the thymus as T-cell progenitors. The rest of the ELPs remain in the bone marrow as B-cell progenitors. Their levels of the interleukin-7 receptor (IL-7R) increase, and the ELP now develops into a CLP, a progenitor that is now c-KitlowSca-1lowIL-7R+ and has lost myeloid potential. However, it still has the potential to mature into any of the lymphocyte lineages: T cell, B cell, or ILC.
FIGURE 2-3 An example of lineage commitment during hematopoiesis: the development of B cells from HSCs. The maturation of HSCs into lymphoid progenitors, and the progressive loss of the ability to differentiate into other blood-cell lineages, is exemplified in this figure, which specifically traces the development of B lymphocytes from multipotent progenitors (MPPs). As cells mature from MPP to lymphoid-primed multipotent progenitors (LMPPs) to common lymphoid progenitors (CLPs) they progressively lose the ability to differentiate into other leukocytes. Pre-pro B cells are committed to becoming B lymphocytes. These changes are also accompanied by changes in expression of cell surface markers as well as by the acquisition of RAG and TdT activity.
Genetic Regulation of Lineage Commitment during Hematopoiesis
Each step a hematopoietic stem cell takes toward commitment to a particular blood cell lineage is accompanied by genetic changes. HSCs maintain a relatively large number of genes in a “primed” state, meaning that they are accessible to transcriptional machinery. Environmental signals that induce HSC differentiation upregulate distinct sets of transcription factors that drive the cell down one of a number of possible developmental pathways. As cells progress down a lineage pathway, primed chromatin regions containing genes that are not needed for the selected developmental pathway are shut down. Many transcription factors that regulate hematopoiesis and lineage choices have been identified. Some have distinct functions, but many are involved at several developmental stages and engage in complex regulatory networks. Some transcription factors associated with hematopoiesis are illustrated in Figure 2-2. However, our understanding of their roles continues to evolve.
A suite of factors appear to regulate HSC quiescence, proliferation, and differentiation (see Figure 2-2). Recent sequencing techniques have identified a “top ten” that include GATA-2, RUNX1, Scl/Tal-1, Lyl1, Lmo2, Meis1, PU.1, ERG, Fli-1, and Gfi1b, although others are bound to play a role. Other transcriptional regulators regulate myeloid versus lymphoid cell lineage choices. For instance, Ikaros is required for lymphoid but not myeloid development; animals survive in its absence but cannot mount a full immune response (i.e., they are severely immunocompromised). Low levels of PU.1 also favor lymphoid differentiation, whereas high levels of PU.1 direct cells to a myeloid fate. Activity of Notch1, one of four Notch family members, induces lymphoid progenitors to develop into T rather than B lymphocytes (see Chapter 8). GATA-1 directs myeloid progenitors toward red blood cell (erythroid) development rather than granulocyte/monocyte lineages. PU.1 also regulates the choice between erythroid and other myeloid cell lineages.
Distinguishing Blood Cells
Historically, investigators classified cells on the basis of their appearance under a microscope, often with the help of dyes. Their observations were especially helpful in distinguishing myeloid from lymphoid lineages, granulocytes from macrophages, and neutrophils from basophils and eosinophils. The pH-sensitive stains hematoxylin and eosin (H&E) are still commonly used in combination to distinguish cell types in blood smears and tissues. The basic dye hematoxylin binds basophilic nucleic acids, staining them blue, and the acidic dye eosin (named for Eos, the goddess of dawn) binds eosinophilic proteins in granules and cytoplasm, staining them pink.
Microscopists drew astute inferences about cell function by detailed examination of stained and unstained cells. Fluorescence microscopy enhanced our ability to identify more molecular details, and in the 1980s, inspired the development of the flow cytometer. This invention revolutionized the study of immunology by allowing us to rapidly measure the presence of multiple surface and internal proteins on individual cells. In vivo cell imaging techniques now permit us to penetrate the complexities of the immune response in time and space. Together with our ever-increasing ability to edit animal and cell genomes, these technologies have revealed an unanticipated diversity of hematopoietic cell types, functions, and interactions. While our understanding of the cell subtypes is impressive, it is by no means complete. Table 2-1 lists the major myeloid and lymphoid cell types, as well as their life spans and representation in our blood.
| Cell type | Cells/mm3 | Total leukocytes (%) | Life span* |
|---|---|---|---|
| Myeloid cells | |||
| Red blood cell | 5.0 × 106 | 120 days | |
| Platelet | 2.5 × 105 | 5–10 days | |
| Neutrophil | 3.7–5.1 × 103 | 50–70 | 6 hours to 2 days |
| Monocyte | 1–4.4 × 102 | 2–12 | Days to months |
| Eosinophil | 1–2.2 × 102 | 1–3 | 5–12 days |
| Basophil | <1.3 × 102 | <1 | Hours to days |
| Mast cell | <1.3 × 102 | <1 | Hours to days |
| Lymphocytes | 1.5–3.0 × 103 | 20–40 | Days to years |
| T lymphocytes | 0.54-1.79 × 103 | 7-24 | |
| B lymphocytes | 0.07-0.53 × 103 | 1-10 | |
| Total leukocytes | 7.3 × 103 | ||
Cells of the Myeloid Lineage Are the First Responders to Infection
Myeloid lineage cells include all red blood cells, granulocytes, monocytes, and macrophages. The white blood cells within this lineage are innate immune cells that respond rapidly to the invasion of a pathogen and communicate the presence of an insult to cells of the lymphoid lineage (below). As we will see in Chapter 15, they also contribute to inflammatory diseases (asthma and allergy).
Granulocytes
Granulocytes are often the first responders during an immune response and fall into four main categories: neutrophils, eosinophils, basophils, and mast cells. All granulocytes have multilobed nuclei that make them visually distinctive and easily distinguishable from lymphocytes, whose nuclei are round. Granulocyte subtypes differ by the staining characteristics of their cytoplasmic granules, membrane-bound vesicles that release their contents in response to pathogens (Figure 2-4). These granules contain a variety of proteins with distinct functions: some damage pathogens directly; some regulate trafficking and activity of other white blood cells, including lymphocytes; and some contribute to the remodeling of tissues at the site of infection. See Table 2-2 for a partial list of granule proteins and their functions.
FIGURE 2-4 Examples of granulocytes. (a) Neutrophils, (b) eosinophils, (c) basophils, and (d) mast cells, shown via (left) hematoxylin and eosin (H&E) stains of blood smears, (middle) scanning electron microscopy (SEM), or (right) as a drawing depicting the typical morphology of the indicated granulocyte. Note differences in the shape of the nucleus and in the number, color, and shape of the cytoplasmic granules.
| Cell type | Molecule in granule | Examples | Function |
|---|---|---|---|
| Neutrophil | Proteases | Elastase, collagenase | Tissue remodeling |
| Antimicrobial proteins | Defensins, lysozyme | Direct harm to pathogens | |
| Protease inhibitors | α1-antitrypsin | Regulation of proteases | |
| Histamine | Vasodilation, inflammation | ||
| Eosinophil | Cationic proteins | EPO | Induces formation of ROS |
| MBP | Vasodilation, basophil degranulation | ||
| Ribonucleases | ECP, EDN | Antiviral activity | |
| Cytokines | IL-4, IL-10, IL-13, TNF-α | Modulation of adaptive immune responses | |
| Chemokines | RANTES, MIP-1α | Attract leukocytes | |
| Basophil/mast cell | Cytokines | IL-4, IL-13 | Modulation of adaptive immune |
| Lipid mediators | Leukotrienes | Regulation of inflammation | |
| Histamine | Vasodilation, smooth muscle activation |
Neutrophils constitute the majority (50% to 70%) of circulating leukocytes (see Figure 2-4a) in adult humans and are much more numerous than eosinophils (1%–3%), basophils (<1%), or mast cells (<1%). After differentiation in the bone marrow, neutrophils are released into the peripheral blood and circulate for 7 to 10 hours before migrating into the tissues, where they have a life span of only a few days. In response to many types of infection, innate immune cells generate inflammatory molecules (e.g., chemokines) that promote the development of neutrophils in the bone marrow. This transient increase in the number of circulating neutrophils is called leukocytosis and is used medically as an indication of infection.
Neutrophils swarm in large numbers to the site of infection in response to inflammatory molecules 
(Video 2-4v). Once in the infected tissue, they phagocytose (engulf) bacteria and secrete a range of proteins that have antimicrobial effects and tissue-remodeling potential. Neutrophils are the main cellular components of pus, where they accumulate at the end of their short lives. Once considered a simple and “disposable” effector cell, neutrophils are now thought to play a regulatory role in shaping the adaptive immune response.
Eosinophils contain granules that stain a brilliant pink in standard H&E staining protocols. They are thought to be important in coordinating our defense against multicellular parasitic organisms, including helminths (parasitic worms). Eosinophils cluster around invading worms, and damage their membranes by releasing the contents of their eosinophilic granules. Like neutrophils, eosinophils are motile cells (see Figure 2-4b) that migrate from the blood into the tissue spaces. They are most abundant in the small intestines, where their role is still being investigated. In areas where parasites are less of a health problem, eosinophils are better appreciated as contributors to asthma and allergy symptoms. Like neutrophils, eosinophils may also secrete cytokines that regulate B and T lymphocytes, thereby influencing the adaptive immune response.
Basophils are nonphagocytic granulocytes (see Figure 2-4c) that contain large basophilic granules that stain blue in standard H&E staining protocols. Basophils are relatively rare in the circulation, but are potent responders. Like eosinophils, basophils are thought to play a role in our response to parasites, particularly helminths (parasitic worms). When they bind circulating antibody/antigen complexes basophils release the contents of their granules. Histamine, one of the best known compounds in basophilic granules, increases blood vessel permeability and smooth muscle activity, and allows immune cells access to a site of infection. Basophils also release cytokines that can recruit other immune cells, including eosinophils and lymphocytes. In areas where parasitic worm infection is less prevalent, histamines are best appreciated as a cause of allergy symptoms.
Mast cells (see Figure 2-4d) also play a role in combating parasitic worms and contribute to allergies. They are released from the bone marrow into the blood as undifferentiated cells. They mature only after they leave the blood for a wide variety of tissues, including the skin, connective tissues of various organs, and mucosal epithelial tissue of the respiratory, genitourinary, and digestive tracts. Like circulating basophils, these cells have large numbers of cytoplasmic granules that contain histamine and other pharmacologically active substances.
Basophils and mast cells share many features, and basophils were once considered the blood-borne version of mast cells. However, recent data suggest that basophils and mast cells have distinct origins and functions.
Myeloid Antigen-Presenting Cells
Myeloid progenitors also give rise to three groups of phagocytic cells—monocytes, macrophages, and dendritic cells—the cells of each of these groups have professional antigen-presenting cell (pAPC) function (Figure 2-5).
FIGURE 2-5 Examples of monocytes, macrophages, dendritic cells, and megakaryocytes. (a) Monocytes, (b) macrophages, (c) dendritic cells, and (d) megakaryocytes, shown via (left) H&E stains of blood smears, (middle) SEM, or (right) as a drawing depicting the typical morphology of the indicated cell. Note that macrophages are five- to tenfold larger than monocytes and contain more organelles, especially lysosomes.
Professional APCs form important cellular bridges between the innate and adaptive immune systems. They become activated after making contact with a pathogen at the site of infection. They communicate this encounter to T lymphocytes in the lymph nodes by displaying peptides from the pathogen to lymphocytes, a process called antigen presentation (discussed in Chapter 7). All cells have the capacity to present peptides from internal proteins using MHC class I molecules; however, pAPCs also have the ability to present peptides from external sources using MHC class II molecules (also discussed in Chapter 7). Only MHC class II molecules can be recognized by helper T cells, which initiate the adaptive immune response. (See “Cells of the Lymphoid Lineage” below and Chapter 7 for more information about MHC molecules.)
Professional APCs exhibit three major activities when they encounter pathogens (and thereby become activated):
- They secrete proteins that attract and activate other immune cells.
- They internalize pathogens via phagocytosis, digest pathogenic proteins into peptides, and then present these peptide antigens on their membrane surfaces via MHC class II molecules.
- They upregulate costimulatory molecules required for optimal activation of helper T cells.
Each variety of pAPC plays a distinct role during the immune response, depending on its locale and its ability to respond to pathogens. Dendritic cells, for example, play a primary role in presenting antigen to—and activating—naïve T lymphocytes (lymphocytes that have not yet been activated by binding antigen). Macrophages are superb phagocytes and are especially efficient at removing both pathogen and damaged host cells from a site of infection. Monocytes regulate inflammatory responses at sites of tissue damage and infection. Investigators have now identified more varieties of APCs than ever anticipated. The functions of these subpopulations are under investigation; some will be described in more detail in coming chapters.
Monocytes constitute from 2% to 12% of white blood cells. They are a heterogeneous group of cells that migrate into tissues and differentiate into a diverse array of tissue-resident phagocytic cells (see Figure 2-5a). Two broad categories of monocytes have been identified. Inflammatory monocytes enter tissues quickly in response to infection. Patrolling monocytes crawl slowly along blood vessels, monitoring their repair. They also provide a reservoir for tissue-resident monocytes in the absence of infection, and may quell rather than initiate immune responses.
Monocytes that migrate into tissues in response to infection can differentiate into macrophages (Figure 2-5b). These inflammatory macrophages are expert phagocytes and typically participate in the innate immune response. They undergo a number of key changes when stimulated by tissue damage or pathogens and have a dual role in the immune response: (1) they contribute directly to the clearance of pathogens from a tissue, and (2) they act as pAPCs for T lymphocytes.
Interestingly, recent work indicates that most tissue-resident macrophages actually arise early in life from embryonic cells rather than from circulating, activated monocytes. These resident macrophages, which include Kupffer cells in the liver, microglia in the brain, and alveolar macrophages in the lungs, have the ability to self-renew and form a committed part of the tissue microenvironment. They co-exist with circulating macrophages and share their function as pAPCs. However, they also assume tissue-specific functions. Table 2-3 includes a more complete list of tissue-resident macrophages and functions.
| Tissue | Name | Tissue-specific function (in addition to activity as pAPCs) |
|---|---|---|
| Brain | Microglia | Neural circuit development (synaptic pruning) |
| Lung | Alveolar macrophage | Remove pollutants and microbes, clear surfactants |
| Liver | Kupffer cell | Scavenge red blood cells, clear particles |
| Kidney | Resident kidney macrophage | Regulate inflammatory responses to antigen filtered from blood |
| Skin | Langerhans cell | Skin immunity and tolerance |
| Spleen | Red pulp macrophage | Scavenge red blood cells, recycle iron |
| Peritoneal cavity | Peritoneal cavity macrophage | Maintain IgA production by B-1 B cells |
| Intestine | Lamina propria macrophage
Intestinal muscularis macrophage |
Gut immunity and tolerance
Regulate peristalsis |
| Bone marrow | Bone marrow macrophage | Maintain niche for blood cell development, clear neutrophils |
| Lymph node | Subcapsular sinus macrophage | Trap antigen particles |
| Heart | Cardiac macrophage | Clear dying heart cells |
Many macrophages express receptors for certain classes of antibody. If a pathogen (e.g., a bacterium) is coated with the appropriate antibody, the complex of antigen and antibody binds to antibody receptors on the macrophage membrane and enhances phagocytosis. In one study, the rate of phagocytosis of an antigen was 4000-fold higher in the presence of specific antibody to the antigen than in its absence. Thus, an antibody is an example of an opsonin, a molecule that binds an antigen and enhances its recognition and ingestion by phagocytes. The modification of antigens with opsonins is called opsonization, a term from the Greek that literally means “to supply food” or “make tasty.” Opsonization serves multiple purposes that will be discussed in subsequent chapters.
Ralph Steinman was awarded the Nobel Prize in Physiology or Medicine in 2011 for his discovery of the dendritic cell (DC) in the mid-1970s. Dendritic cells (Figure 2-5c) are critical for the initiation of the immune response and acquired their name because they extend and retract long membranous extensions that resemble the dendrites of nerve cells. These processes increase the surface area available for browsing lymphocytes. Dendritic cells are a more diverse population of cells than once was thought, and seem to arise from both the myeloid and lymphoid lineages of hematopoietic cells. The functional distinctions among dendritic cell populations are still being clarified, and each subtype is likely critically important in tailoring immune responses to distinct pathogens and targeting responding cells to distinct tissues.
Dendritic cells perform the distinct functions of antigen capture in one location and antigen presentation in another. Outside lymph nodes, immature forms of these cells monitor the body for signs of invasion by pathogens and capture intruding or foreign antigens. They process these antigens and migrate to lymph nodes, where they present the antigen to naïve T cells, initiating the adaptive immune response.
When acting as sentinels in the periphery, immature dendritic cells take in antigen in three ways. They engulf it by phagocytosis, internalize it by receptor-mediated endocytosis, or imbibe it by pinocytosis. Indeed, immature dendritic cells pinocytose fluid volumes of 1000 to 1500 mm3 per hour, a volume that rivals that of the cell itself. After antigen contact, they mature from an antigen-capturing phenotype to one that is specialized for presentation of antigen to T cells. In making this transition, some attributes are lost and others are gained. Dendritic cells that have captured antigen lose the capacity for phagocytosis and large-scale pinocytosis. They improve their ability to present antigen and express costimulatory molecules essential for the activation of naïve T cells. After maturation, dendritic cells enter the blood or lymphatic circulation, and migrate to regions containing lymphoid organs, where they present antigen to circulating T cells.
It is important to note that follicular dendritic cells (FDCs) do not arise from hematopoietic stem cells and are functionally distinct from dendritic cells. FDCs were named not only for their dendrite-like processes, but for their exclusive location in follicles, organized structures in secondary lymphoid tissue that are rich in B cells. Unlike dendritic cells, FDCs are not pAPCs and do not activate naïve T cells. Instead, they regulate the activation of B cells, as discussed in Chapters 11 and 14.
Erythroid Cells
Cells of the erythroid lineage—erythrocytes, or red blood cells—also arise from myeloid progenitors. Erythrocytes contain high concentrations of hemoglobin, and circulate through blood vessels and capillaries delivering oxygen to surrounding cells and tissues. Damaged red blood cells also release signals that induce innate immune activity. In mammals, erythrocytes are anuclear; their nucleated precursors, erythroblasts, extrude their nuclei in the bone marrow. However, the erythrocytes of nonmammalian vertebrates (birds, fish, amphibians, and reptiles) retain their nuclei. Erythrocyte size and shape vary considerably across the animal kingdom—the largest red blood cells can be found among some amphibians, and the smallest among some deer species.
Although the main function of erythrocytes is gas exchange, they may also play a more direct role in immunity. They express surface receptors for antibody and bind antibody complexes that can then be cleared by the many macrophages that scavenge erythrocytes. They also generate compounds, like nitric oxide (NO), that do direct damage to microbes.
Megakaryocytes
Megakaryocytes are large myeloid cells that reside in the bone marrow and give rise to thousands of platelets, very small cells (or cell fragments) that circulate in the blood and participate in the formation of blood clots (Figure 2-5d). Clots not only prevent blood loss, but when they take place at epithelial barriers, they also provide a barrier against the invasion of pathogens. Although platelets have some of the properties of independent cells, they do not have their own nuclei.
Cells of the Lymphoid Lineage Regulate the Adaptive Immune Response
Lymphoid lineage cells, or lymphocytes (Figure 2-6), are the principal cell players in the adaptive immune response and the source of immune memory. They represent 20% to 40% of circulating white blood cells and 99% of cells in the lymph. Lymphocytes are broadly subdivided into three major populations on the basis of functional and phenotypic differences: B lymphocytes (B cells), T lymphocytes (T cells), and innate lymphoid cells (ILCs), which include the well-understood natural killer (NK) cells. In humans, approximately a trillion (1012) lymphocytes circulate continuously through the blood and lymph and migrate into the tissue spaces and lymphoid organs. Large numbers of lymphocytes reside in the tissues that line our intestines, airways, and reproductive tracts, too. We briefly review the general characteristics and functions of each lymphocyte group and its subsets below.
FIGURE 2-6 Examples of lymphocytes. (a) H&E stain of a blood smear showing a typical lymphocyte, with drawings depicting naïve TH, TC, and B cells. Note that naïve B cells and T cells look identical by microscopy. (b) SEM of a lymphocyte with red blood cells. (c) H&E stain of a blood smear showing a plasma cell, with a drawing depicting the plasma cell’s typical morphology. The cytoplasm of the plasma cell is enlarged because it is occupied by an extensive endoplasmic reticulum network—an indication of the cell’s dedication to antibody production. (d) H&E stain of a blood smear showing an innate lymphoid cell, the natural killer (NK) cell. NK cells have more cytoplasm than a naïve lymphocyte; this one is full of granules that are used to kill target cells. (e) A branch diagram that depicts the basic relationship among the lymphocyte subsets described in the text. (Abbreviations: CLP = common lymphoid progenitor; ILC = innate lymphoid cell; TH1, TH2, TH17 = helper T type 1, 2, and 17 cells, respectively; TREG = regulatory T cell.)
Small, round, and dominated by their nucleus, lymphocytes are relatively nondescript cells. T and B lymphocytes, in fact, appear identical under a microscope. We therefore rely heavily on the profile of surface proteins they express to differentiate lymphocyte subpopulations.
Surface proteins expressed by cells of the immune system (as well as some other cells) are often referred to by the cluster of differentiation (CD) nomenclature. This nomenclature was established in 1982 by an international group of investigators who recognized that many of the new antibodies produced by laboratories all over the world (largely in response to the advent of monoclonal antibody technology) were binding to the same proteins, and hence some proteins were given multiple names by different labs. The group therefore defined clusters of antibodies that appeared to be binding to the same protein and assigned a name—a cluster of differentiation or CD—to each protein. Although originally designed to categorize the multiple antibodies, the CD nomenclature is now firmly associated with specific surface proteins found on cells of many types. Table 2-4 lists some common CD molecules found on human and mouse lymphocytes. Note that the shift from use of a “common” name to the more standard “CD” name has taken place slowly. For example, investigators often still refer to the pan-T cell marker as “Thy-1” rather than CD90, and the costimulatory molecules as “B7-1” and “B7-2,” rather than CD80 and CD86. Appendix I lists over 300 CD markers expressed by immune cells.
| CD designation | Function | B cell | TH cell | TC cell | NK cell1 |
|---|---|---|---|---|---|
| CD2 | Adhesion molecule; signal transduction | – | + | + | + |
| CD3 | Signal transduction element of T-cell receptor | – | + | + | – |
| CD4 | Adhesion molecule that binds to MHC class II molecules; signal transduction | – | + (usually) | – (usually) | – |
| CD5 | Unknown | +(subset) | + | + | + |
| CD8 | Adhesion molecule that binds to MHC class I molecules; signal transduction | – | – (usually) | + (usually) | Variable |
| CD16 (FcγRIII) | Low-affinity receptor for Fc region of IgG | – | – | – | + |
| CD19 | Signal transduction; CD21 coreceptor | + | – | – | – |
| CD20 | Signal transduction; regulates Ca2+ transport across the membrane | + | – | – | – |
| CD21 (CR2) | Receptor for complement (C3d) and Epstein–Barr virus | + | – | – | – |
| CD28 | Receptor for costimulatory B7 molecule on antigen-presenting cells | – | + | + | – |
| CD32 (FcγRII) | Receptor for Fc region of IgG | + | – | – | – |
| CD35 (CR1) | Receptor for complement (C3b) | + | – | – | – |
| CD40 | Signal transduction | + | – | – | – |
| CD45 | Signal transduction | + | + | + | + |
| CD56 | Adhesion molecule | – | – | – | + |
| CD161 (NK1.1) | Lectin-like receptor | – | – | – | + |
B and T cells express many different CD proteins on their surface, depending on their stage of development and state of activation. In addition, each B or T cell also expresses an antigen-specific receptor (the B-cell receptor or the T-cell receptor, respectively) on its surface. Although B and T cell populations express a remarkable diversity of antigen receptors (more than a billion), all antigen-specific receptors on an individual cell’s surface are identical in structure and, therefore, are identical in their specificity for antigen. When a particular T or B cell divides, all of its progeny will also express this specific antigen receptor. The resulting population of lymphocytes, all arising from the same founding lymphocyte, is a clone (see Figure 1-6).
At any given moment, tens of thousands, perhaps a hundred thousand, distinct mature T- and B-cell clones circulate in a human or mouse, each distinguished by its unique antigen receptor. Newly formed B cells and T cells are considered naïve. Contact with antigen induces naïve lymphocytes to proliferate and differentiate into both effector cells and memory cells. Effector cells carry out specific functions to combat the pathogen, while memory cells persist in the host, and when rechallenged with the same antigen, respond faster and more efficiently. As you have learned in Chapter 1, the first encounter with antigen generates a primary response, and the re-encounter a secondary response (see Figure 1-8).
B Lymphocytes
The B lymphocyte (B cell) derived its letter designation from its site of maturation, in the bursa of Fabricius in birds; the name turned out to be apt, as bone marrow is its major site of maturation in humans, mice, and many other mammals. Mature B cells are definitively distinguished from other lymphocytes and all other cells by their expression of the B-cell receptor (BCR), a membrane-bound immunoglobulin (antibody) molecule that binds to antigen (see Figure 2-7a and Chapter 3). Each B cell expresses a surface antibody with a unique specificity, and each of the approximately 1.5–3 × 105 molecules of surface antibody on a B cell has identical binding sites for antigen. B lymphocytes also improve their ability to bind antigen through a process known as somatic hypermutation and can generate antibodies of several different functional classes through a process known as class switching. Somatic hypermutation and class switching are covered in detail in Chapter 11.
FIGURE 2-7 Structure of the B-cell and T-cell antigen receptors. (a) B-cell receptors recognize soluble antigens. (b) T-cell receptors (TCRs) recognize membrane-bound MHC–peptide complexes. This figure shows a TCR from a helper T cell that recognizes MHC class II (with the assistance of the CD4 molecule). A TCR from a cytotoxic T cell (not shown) would recognize MHC class I with the assistance of a CD8 molecule. (Abbreviations: APC = antigen-presenting cell; BCR = B-cell receptor; MHC = major histocompatibility complex.)
Activated B lymphocytes are the only nonmyeloid cell that can act as a pAPC. They internalize antigen very efficiently via their antigen-specific receptor, and process and present antigenic peptides at the cell surface. Activated B cells also express costimulatory molecules required to activate T cells. By presenting antigen directly to T cells, B cells also receive T-cell help, in the form of cytokines that induce their differentiation into antibody-producing cells (plasma cells) and memory cells.
Ultimately, activated B cells differentiate into effector cells known as plasma cells (see Figure 2-6c). Plasma cells lose expression of surface immunoglobulin and become highly specialized for secretion of antibody. A single cell is capable of secreting from a few hundred to more than a thousand molecules of antibody per second. Plasma cells do not divide and, although some travel to the bone marrow and live for years, others die within 1 or 2 weeks.
T Lymphocytes
T lymphocytes (T cells) derive their letter designation from their site of maturation in the thymus. Like the B cell, the T cell expresses a unique antigen-binding receptor called the T-cell receptor (TCR; see Figure 2-7b and Chapter 3). However, unlike membrane-bound antibodies on B cells, which can recognize soluble or particulate antigen, T-cell receptors recognize only processed pieces of antigen (typically peptides) bound to cell membrane proteins called major histocompatibility complex (MHC) molecules. MHC molecules are genetically diverse glycoproteins found on cell membranes. They were identified as the cause of rejection of transplanted tissue, and their structure and function are covered in detail in Chapter 7. The ability of MHC molecules to form complexes with antigen allows cells to decorate their surfaces with internal (foreign and self) proteins, exposing them to browsing T cells. MHC comes in two versions: MHC class I molecules, which are expressed by nearly all nucleated cells of vertebrate species, and MHC class II molecules, which are expressed primarily by pAPCs.
T lymphocytes are divided into two major cell types—T helper (TH) cells and T cytotoxic (TC) cells—that can be distinguished from one another by the presence of either CD4 or CD8 membrane glycoproteins on their surfaces. T cells displaying CD4 generally function as helper (TH) cells and recognize antigen in complex with MHC class II, whereas those displaying CD8 generally function as cytotoxic (TC) cells and recognize antigen in complex with MHC class I (see Figure 2-8 and Chapter 12). The ratio of CD4+ to CD8+ T cells is approximately 2:1 in healthy mouse and human peripheral blood. A change in this ratio is often an indication of immunodeficiency disease (e.g., HIV infection), autoimmune disease, aging, and inflammation.
FIGURE 2-8 T-cell recognition of antigen. T cells displaying CD4 generally function as helper (TH) cells and recognize peptide antigen associated with MHC class II, whereas those displaying CD8 generally function as cytotoxic (TC) cells and recognize peptide antigen associated with MHC class I.
Naïve CD8+ TC cells browse the surfaces of antigen-presenting cells with their T-cell receptors. If and when they bind to an MHC-peptide complex, they become activated, proliferate, and differentiate into a type of effector cell called a cytotoxic T lymphocyte (CTL). The CTL has a vital function in monitoring the cells of the body and eliminating cells that display non-self-antigen complexed with MHC class I, such as virus-infected cells, tumor cells, and cells of a foreign tissue graft. To proliferate and differentiate optimally, naïve CD8+ T cells also need help from mature CD4+ T cells.
Naïve CD4+ TH cells also browse the surfaces of antigen-presenting cells with their T-cell receptors. If and when they recognize an MHC-peptide complex, they become activated and proliferate and differentiate into one of a variety of effector T-cell subsets (see Figure 2-6e). In broad terms, T helper type 1 (TH1) cells and T helper type 17 (TH17) cells (the latter so named because they secrete IL-17) regulate our response to intracellular pathogens, and T helper type 2 (TH2) cells and T follicular helper (TFH) cells regulate our response to extracellular pathogens, such as bacteria and parasitic worms. Each CD4+ TH-cell subtype produces a different set of cytokines that enable or “help” the activation of B cells, Tc cells, macrophages, and various other cells that participate in the immune response.
Which helper subtype dominates a response depends largely on what type of pathogen (intracellular versus extracellular, viral, bacterial, fungal, helminth) has infected an animal. The network of cytokines that regulate and are produced by these effector cells is described in detail in Chapter 10.
Another type of CD4+T cell, the regulatory T cell (TREG), has the unique capacity to inhibit immune responses. These cells, called natural TREG cells, arise during maturation in the thymus from cells that bind self proteins with high affinity (autoreactive cells). They can also can be induced at the site of an immune response in an antigen-dependent manner (iTREG cells). Regulatory T cells are identified by the presence of CD4 and CD25 on their surfaces, as well as by the expression of the internal transcription factor FoxP3. TREG cells quell autoreactive responses and play a role in limiting our normal T-cell responses to pathogens.
Both CD4+ and CD8+ T-cell subpopulations may be even more diverse than currently described, and additional functional subtypes could be identified in the future.
NKT Cells
Another type of cell in the lymphoid lineage, the NKT cell, shares features with both adaptive and innate immune cells. Like T cells, NKT cells have T-cell receptors (TCRs), and some express CD4. Unlike most T cells, however, the TCRs of NKT cells are not diverse. Rather than recognize protein peptides, they recognize specific lipids and glycolipids presented by a molecule related to MHC proteins known as CD1. NKT cells also have receptors classically associated with innate immune cells, including the NK cells discussed below. Activated NKT cells release cytotoxic granules that kill target cells, but also release large quantities of cytokines that can both enhance and suppress the immune response. They appear to be involved in human asthma, but also may inhibit the development of autoimmunity and cancer. Understanding the exact role of NKT cells in immunity is one research priority.
Innate Lymphoid Cells (ILCs)
Investigators now recognize a group of cells that are derived from common lymphoid progenitors, but do not express antigen-specific receptors: innate lymphoid cells (ILCs). Currently they are subdivided into three groups (ILC1, ILC2, and ILC3), distinguished by the cytokines they secrete, which mirror those produced by distinct helper T-cell subsets (Table 2-5). Many provide a first line of defense against pathogens in the skin and at mucosal tissues (Chapter 13). ILC helper subsets are the focus of active investigation, and the nomenclature describing ILC subtypes is still in flux as investigators work to determine their origins and their relationships to each other and to other blood cells.
| ILC | T-cell subset | Signature cytokines secreted by both | Master transcriptional regulators of both |
|---|---|---|---|
| Group 1 ILCs | |||
| NK cell | CTL | IFN-γ, perforin, granzyme | T-bet |
| ILC1 | TH1 | IFN-γ, TNF | |
| Group 2 ILCs | |||
| ILC2 | TH2 | IL-4, IL-5, IL-13, and amphiregulin | GATA-3 |
| Group 3 ILCs | |||
| LTi cell | TH17, TH22 | IL-17A, IL-22, LTα, LTβ, | RORγt |
| ILC3 | IL-22, IFN-γ | ||
Cytotoxic natural killer (NK) cells are the founding members of the innate lymphoid cell category and the best studied. Many investigators classify NK cells within the ILC1 group; some define them as a distinct cytotoxic lineage of ILCs. Regardless of their classification, NK cells are identified by their expression of the NK1.1 surface protein and constitute 5% to 10% of lymphocytes in human peripheral blood.
Efficient cell killers, they use two different strategies to attack a variety of abnormal cells. The first strategy is to attack cells that lack MHC class I molecules. Infection by certain viruses or mutations occurring in tumor cells often cause those cells to downregulate MHC class I. NK cells express a variety of receptors for self-MHC class I that, when engaged, inhibit their ability to kill. However, when NK cells encounter cells that have lost their MHC class I, these inhibiting receptors are no longer engaged and NK cells can release their cytotoxic granules, killing the target cell.
Second, NK cells express receptors (called Fc receptors or FcRs) for some antibodies. By linking these receptors to antibodies, NK cells can arm themselves with antibodies specific for pathogenic proteins, particularly viral proteins present on the surfaces of infected cells. Once such antibodies bring the NK cell in contact with target cells, the NK cell releases its granules and induces cell death, a process known as antibody-dependent cell cytotoxicity (ADCC). The mechanisms of NK-cell cytotoxicity are described further in Chapter 12.
Our understanding of ILCs is still in its infancy, and ongoing investigations are continually generating new insights into their origin and function.