Lymphocytes constitute 20%-40% of the body's white blood cells and 99% of the cells in the lymph (Table 2-4). There are approximately 1011 (range depending on body size and age: ~101o-1012) lymphocytes in the human body. These lymphocytes continually circulate in the blood and lymph and are capable of migrating into the tissue spaces and lymphoid organs, thereby integrating the immune system to a high degree.
The lymphocytes can be broadly subdivided into three populations—B cells, T cells, and natural killer cells—on the basis of function and cell-membrane components. Natural killer cells (NK cells) are large, granular lymphocytes that do not express the set of surface markers typical of B or T cells. Resting B and T lymphocytes are small, motile, nonphago-cytic cells, which cannot be distinguished morphologically. B and T lymphocytes that have not interacted with antigen— referred to as naive, or unprimed—are resting cells in the G0 phase of the cell cycle. Known as small lymphocytes, these cells are only about 6 ^m in diameter; their cytoplasm forms a barely discernible rim around the nucleus. Small lymphocytes have densely packed chromatin, few mitochondria, and a poorly developed endoplasmic reticulum and Golgi apparatus. The naive lymphocyte is generally thought to have a short life span. Interaction of small lymphocytes with antigen, in the presence of certain cytokines discussed later, induces these cells to enter the cell cycle by progressing from G0 into G1 and subsequently into S, G2, and M (Figure 2-7a). As they progress through the cell cycle, lymphocytes enlarge into 15 ^m-diameter blast cells, called lymphoblasts; these cells have a higher cytoplasm:nucleus ratio and more organellar complexity than small lymphocytes (Figure 2-7b).
Lymphoblasts proliferate and eventually differentiate into effector cells or into memory cells. Effector cells function in various ways to eliminate antigen. These cells have short life
TABLE 2-4l Normal adult blood-cell counts
Cell type Cells/mm3 %
Red blood cells 5.0 X 10s
Platelets 2.5 X 105
Leukocytes 7.3 X 103
Go to www.whfreeman.com/immunology i Animation Cells and Organs of the Immune System
Small lymphocyte (T or B) 6 |im diameter
Blast cell (T or B) 15 |im diameter
Plasma cell (B) 15 |im diameter
Fate of antigen-activated small lymphocytes. (a) A small resting (naive or unprimed) lymphocyte resides in the G0 phase of the cell cycle. At this stage, B and T lymphocytes cannot be distinguished morphologically. After antigen activation, a B or T cell enters the cell cycle and enlarges into a lymphoblast, which undergoes several rounds of cell division and, eventually, generates effector cells and memory cells. Shown here are cells of the B-cell lineage. (b) Electron micrographs of a small lymphocyte (left) showing con densed chromatin indicative of a resting cell, an enlarged lymphoblast (center) showing decondensed chromatin, and a plasma cell (right) showing abundant endoplasmic reticulum arranged in concentric circles and a prominent nucleus that has been pushed to a characteristically eccentric position. The three cells are shown at different magnifications. [Micrographs courtesy of Dr. J. R. Goodman, Dept. of Pediatrics, University of California at San Francisco.]
CLI N ICAL FOCUS
CLI N ICAL FOCUS
Stem Cells—Clinical Uses and Potential
Stem-cell transplantation holds great promise for the regeneration of diseased, damaged, or defective tissue. Hematopoietic stem cells are already used to restore hematopoietic cells, and their use is described in the clinic below. However, rapid advances in stem-cell research have raised the possibility that other stem-cell types, too, may soon be routinely employed for replacement of other cells and tissues. Two properties of stem cells underlie their utility and promise. They have the capacity to give rise to more differentiated cells, and they are self-renewing, because each division of a stem cell creates at least one stem cell. If stem cells are classified according to their descent and developmental potential, four levels of stem cells can be recognized: totipotent, pluripotent, multipotent, and unipotent.
Totipotent cells can give rise to an entire organism. A fertilized egg, the zygote, is a totipotent cell. In humans the initial divisions of the zygote and its descendants produce cells that are also totipotent. In fact, identical twins, each with its own placenta, develop when totipotent cells separate and develop into genetically identical fetuses. Pluripotent stem cells arise from totipotent cells and can give rise to most but not all of the cell types necessary for fetal development. For example, human pluripotent stem cells can give rise to all of the cells of the body but cannot generate a placenta. Further differentiation ofpluripo-tent stem cells leads to the formation of multipotent and unipotent stem cells. Multipotent stem cells can give rise to only a limited number of cell types, and unipotent cells to a single cell type. Pluripotent cells, called embryonic stem cells, or simply ES cells, can be isolated from early embryos, and for many years it has been possible to grow mouse ES cells as cell lines in the laboratory. Strikingly, these ES cells can be induced to generate many different types of cells. Mouse ES cells have been shown to give rise to muscle cells, nerve cells, liver cells, pancreatic cells, and, of course, hematopoietic cells.
Recent advances have made it possible to grow lines of human pluripotent cells. This is a development of considerable importance to the understanding of human development, and it has great therapeutic potential. In vitro studies of the factors that determine or influence the development of human pluripotent stem cells along one developmental path as opposed to another will provide considerable insight into the factors that affect the differentiation of cells into specialized types. There is also great interest in exploring the use of pluripotent stem cells to generate cells and tissues that could be used to replace diseased or damaged ones. Success in this endeavor would be a major advance because transplantation medicine now depends totally upon donated organs and tissues. Unfortunately, the need far exceeds the number of donations and is increasing. Success in deriving practical quantities of cells, tissues, and organs from pluripotent stem cells would provide skin replacement for burn patients, heart muscle cells for those with chronic heart disease, pancreatic islet cells for patients with diabetes, and neurons for use in Parkinson's disease or Alzheimer's disease.
The transplantation of hematopoietic stem cells (HSCs) is an important therapy for patients whose hematopoietic systems must be replaced. It has three major applications:
1. Providing a functional immune system to individuals with a genetically determined immunodeficiency, such as severe
Heart muscle cells Pancreatic islet cells
Human pluripotent stem cells can differentiate into a variety of different cell types, some of which are shown here. [Adapted from Stem Cells: A Primer, NIH web site http://www.nih.gov/news/stemcell/primer.htm. Micrographs (left to right): Biophoto Associates/Science Source/Photo Researchers; Biophoto Associates/Photo Researchers; AFIP/Science Source/Photo Researchers; Astrid & Hanns-Frieder Michler/Science Photo Library/Photo Researchers.]
combined immunodeficiency (SCID).
2. Replacing a defective hematopoietic system with a functional one to cure some patients who have a life-threatening nonmalignant genetic disorder in hematopoiesis, such as sickle-cell anemia or thalassemia.
3. Restoring the hematopoietic system of cancer patients after treatment with doses of chemotherapeutic agents and radiation so high that they destroy the system. These high-dose regimens can be much more effective at killing tumor cells than are therapies that use more conventional doses of cytotoxic agents. Stem-cell transplantation makes it possible to recover from such drastic treatment. Also, certain cancers, such as some cases of acute myeloid leukemia, can be cured only by destroying the source of the leukemia cells, the patient's own hematopoietic system.
Restoration of the hematopoietic system by transplanting stem cells is facilitated by several important technical considerations. First, HSCs have extraordinary powers of regeneration. Experiments in mice indicate that only a few—perhaps, on occasion, a single HSC—can completely restore the erythroid population and the immune system. In humans it is necessary to administer as little as 10% of a donor's total volume of bone marrow to provide enough HSCs to completely restore the hematopoietic system. Once injected into a vein, HSCs enter the circulation and find their own way to the bone marrow, where they begin the process of engraftment. There is no need for a surgeon to directly inject the cells into bones. In addition, HSCs can be preserved by freezing. This means that hematopoietic cells can be "banked." After collection, the cells are treated with a cryopreservative, frozen, and then stored for later use. When needed, the frozen preparation is thawed and infused into the patient, where it reconstitutes the hematopoietic system. This cell-freezing technology even makes it pos sible for individuals to store their own hematopoietic cells for transplantation to themselves at a later time. Currently, this procedure is used to allow cancer patients to donate cells before undergoing chemotherapy and radiation treatments and then to reconstitute their hematopoietic system from their own stem cells. Hematopoietic stem cells are found in cell populations that display distinctive surface antigens. One of these antigens is CD34, which is present on only a small percentage (-1%) of the cells in adult bone marrow. An antibody specific for CD34 is used to select cells displaying this antigen, producing a population enriched in CD34+ stem cells. Various versions ofthis selection procedure have been used to enrich populations of stem cells from a variety of sources.
Transplantation of stem cell populations may be autologous (the recipient is also the donor), syngeneic (the donor is genetically identical, i.e., an identical twin of the recipient), or allogeneic (the donor and recipient are not genetically identical). In any transplantation procedure, genetic differences between donor and recipient can lead to immune-based rejection reactions. Aside from host rejection of transplanted tissue (host versus graft), lymphocytes in the graft can attack the recipient's tissues, thereby causing graft-versus-host disease (GVHD), a life-threatening affliction. In order to suppress rejection reactions, powerful immunosup-pressive drugs must be used. Unfortunately, these drugs have serious side effects, and immunosuppression increases the patient's risk of infection and further growth of tumors. Consequently, HSC transplantation has fewest complications when there is genetic identity between donor and recipient.
At one time, bone-marrow transplantation was the only way to restore the hematopoietic system. However, the essential element of bone-marrow transplantation is really stem-cell transplantation. Fortunately, significant numbers of stem cells can be obtained from other tissues, such as peripheral blood and umbilical-cord blood ("cord blood"). These alternative sources of HSCs are attractive because the donor does not have to undergo anesthesia and the subsequent highly invasive procedure that extracts bone marrow. Many in the transplantation community believe that peripheral blood will replace marrow as the major source of hematopoietic stem cells for many applications. To obtain HSC-en-riched preparations from peripheral blood, agents are used to induce increased numbers ofcirculating HSCs, and then the HSC-containing fraction is separated from the plasma and red blood cells in a process called leukopheresis. If necessary, further purification can be done to remove T cells and to enrich the CD34+ population.
Umbilical cord blood already contains a significant number of hematopoietic stem cells. Furthermore, it is obtained from placental tissue (the "afterbirth") which is normally discarded. Consequently, umbilical cord blood has become an attractive source of cells for HSC transplantation. Although HSCs from cord blood fail to engraft somewhat more often than do cells from peripheral blood, grafts of cord blood cells produce GVHD less frequently than do marrow grafts, probably because cord blood has fewer mature T cells.
Beyond its current applications in cancer treatment, many researchers feel that autologous stem-cell transplantation will be useful for gene therapy, the introduction of a normal gene to correct a disorder caused by a defective gene. Rapid advances in genetic engineering may soon make gene therapy a realistic treatment for genetic disorders of blood cells, and hematopoietic stem cells are attractive vehicles for such an approach. The therapy would entail removing a sample of hematopoietic stem cells from a patient, inserting a functional gene to compensate for the defective one, and then reinjecting the engineered stem cells into the donor. The advantage of using stem cells in gene therapy is that they are self renewing. Consequently, at least in theory, patients would have to receive only a single injection ofen-gineered stem cells. In contrast, gene therapy with engineered mature lymphocytes or other blood cells would require periodic injections because these cells are not capable of self renewal.
spans, generally ranging from a few days to a few weeks. Plasma cells—the antibody-secreting effector cells of the B-cell lineage—have a characteristic cytoplasm that contains abundant endoplasmic reticulum (to support their high rate of protein synthesis) arranged in concentric layers and also many Golgi vesicles (see Figure 2-7). The effector cells of the T-cell lineage include the cytokine-secreting T helper cell (Th cell) and the T cytotoxic lymphocyte (TC cell). Some of the progeny of B and T lymphoblasts differentiate into memory cells. The persistence of this population of cells is responsible for life-long immunity to many pathogens. Memory cells look like small lymphocytes but can be distinguished from naive cells by the presence or absence of certain cellmembrane molecules.
Different lineages or maturational stages of lymphocytes can be distinguished by their expression of membrane molecules recognized by particular monoclonal antibodies (antibodies that are specific for a single epitope of an antigen; see Chapter 4 for a description of monoclonal antibodies). All of the monoclonal antibodies that react with a particular membrane molecule are grouped together as a cluster of differentiation (CD). Each new monoclonal antibody that recognizes a leukocyte membrane molecule is analyzed for whether it falls within a recognized CD designation; if it does not, it is given a new CD designation reflecting a new membrane molecule. Although the CD nomenclature was originally developed for the membrane molecules of human leukocytes, the homologous membrane molecules of other species, such as mice, are commonly referred to by the same CD designations. Table 2-5 lists some common CD molecules (often referred to as CD markers) found on human lymphocytes. However, this is only a partial listing of the more than 200 CD markers that have been described. A complete list and description of known CD markers is in the appendix at the end of this book.
The general characteristics and functions of B and T lymphocytes were described in Chapter 1 and are reviewed briefly in the next sections. These central cells of the immune system will be examined in more detail in later chapters.
The B lymphocyte derived its letter designation from its site of maturation, in the bursa of Fabricius in birds; the name turned out to be apt, for bone marrow is its major site of maturation in a number of mammalian species, including humans and mice. Mature B cells are definitively distinguished from other lymphocytes by their synthesis and display of membrane-bound immunoglobulin (antibody) molecules,
Common CD markers used to distinguish functional lymphocyte subpopulations
CD2 Adhesion molecule; signal transduction
CD3 Signal-transduction element of T-cell receptor
CD4 Adhesion molecule that binds to class II
MHC molecules; signal transduction
CD8 Adhesion molecule that binds to class I
MHC molecules; signal transduction
CD16 (Fc^RIII) Low-affinity receptor for Fc region of IgG
CD21 (CR2) Receptor for complement (C3d) and
CD28 Receptor for co-stimulatory B7 molecule on antigen-presenting cells
CD32 (Fc^RII) Receptor for Fc region of IgG
CD35 (CR1) Receptor for complement (C3b)
CD40 Signal transduction
CD45 Signal transduction
CD56 Adhesion molecule
"Synonyms are shown in parentheses.
which serve as receptors for antigen. Each of the approximately 1.5 X 105 molecules of antibody on the membrane of a single B cell has an identical binding site for antigen. Among the other molecules expressed on the membrane of mature B cells are the following:
■ B220 (a form of CD45) is frequently used as a marker for B cells and their precursors. However, unlike antibody, it is not expressed uniquely by B-lineage cells.
■ Class II MHC molecules permit the B cell to function as an antigen-presenting cell (APC).
■ CR1 (CD35) and CR2 (CD21) are receptors for certain complement products.
■ FcyRII (CD32) is a receptor for IgG, a type of antibody.
■ B7-1 (CD80) and B7-2 (CD86) are molecules that interact with CD28 and CTLA-4, important regulatory molecules on the surface of different types of T cells, including TH cells.
■ CD40 is a molecule that interacts with CD40 ligand on the surface of helper T cells. In most cases this interaction is critical for the survival of antigen-stimulated B cells and for their development into antibody-secreting plasma cells or memory B cells.
Interaction between antigen and the membrane-bound antibody on a mature naive B cell, as well as interactions with T cells and macrophages, selectively induces the activation and differentiation of B-cell clones of corresponding specificity. In this process, the B cell divides repeatedly and differentiates over a 4- to 5-day period, generating a population of plasma cells and memory cells. Plasma cells, which have lower levels of membrane-bound antibody than B cells, synthesize and secrete antibody. All clonal progeny from a given B cell secrete antibody molecules with the same antigen-binding specificity. Plasma cells are terminally differentiated cells, and many die in 1 or 2 weeks.
T lymphocytes derive their name from their site of maturation in the ihymus. Like B lymphocytes, these cells have membrane receptors for antigen. Although the antigen-binding T-cell receptor is structurally distinct from im-munoglobulin, it does share some common structural features with the immunoglobulin molecule, most notably in the structure of its antigen-binding site. Unlike the membrane-bound antibody on B cells, though, the T-cell receptor (TCR) does not recognize free antigen. Instead the TCR recognizes only antigen that is bound to particular classes of self-molecules. Most T cells recognize antigen only when it is bound to a self-molecule encoded by genes within the major histocompatibility complex (MHC). Thus, as explained in Chapter 1, a fundamental difference between the humoral and cell-mediated branches of the immune system is that the B cell is capable of binding soluble antigen, whereas the T cell is restricted to binding antigen displayed on self-cells. To be recognized by most T cells, this antigen must be displayed together with MHC molecules on the surface of antigen-presenting cells or on virus-infected cells, cancer cells, and grafts. The T-cell system has developed to eliminate these altered self-cells, which pose a threat to the normal functioning of the body.
Like B cells, T cells express distinctive membrane molecules. All T-cell subpopulations express the T-cell receptor, a complex of polypeptides that includes CD3; and most can be distinguished by the presence of one or the other of two membrane molecules, CD4 and CD8. In addition, most mature T cells express the following membrane molecules:
■ CD28, a receptor for the co-stimulatory B7 family of molecules present on B cells and other antigen-
■ CD45, a signal-transduction molecule
T cells that express the membrane glycoprotein molecule CD4 are restricted to recognizing antigen bound to class II MHC molecules, whereas T cells expressing CD8, a dimeric membrane glycoprotein, are restricted to recognition of antigen bound to class I MHC molecules. Thus the expression of CD4 versus CD8 corresponds to the MHC restriction of the T cell. In general, expression of CD4 and of CD8 also defines two major functional subpopulations of T lymphocytes. CD4+ T cells generally function as T helper (TH) cells and are class-II restricted; CD8+ T cells generally function as T cytotoxic (TC) cells and are class-I restricted. Thus the ratio of TH to TC cells in a sample can be approximated by assaying the number of CD4+ and CD8+ T cells. This ratio is approximately 2:1 in normal human peripheral blood, but it may be significantly altered by immunodeficiency diseases, autoimmune diseases, and other disorders.
The classification of CD4+ class II-restricted cells as TH cells and CD8+ class I-restricted cells as TC cells is not absolute. Some CD4+ cells can act as killer cells. Also, some TC cells have been shown to secrete a variety of cytokines and exert an effect on other cells comparable to that exerted by TH cells. The distinction between TH and TC cells, then, is not always clear; there can be ambiguous functional activities. However, because these ambiguities are the exception and not the rule, the generalization of T helper (TH) cells as being CD4+ and class-II restricted and of T cytotoxic cells (TC) as being CD8+ and class-I restricted is assumed throughout this text, unless otherwise specified.
Th cells are activated by recognition of an antigen-class II MHC complex on an antigen-presenting cell. After activation, the TH cell begins to divide and gives rise to a clone of effector cells, each specific for the same antigen-class II MHC complex. These TH cells secrete various cytokines, which play a central role in the activation of B cells, T cells, and other cells that participate in the immune response. Changes in the pattern of cytokines produced by TH cells can change the type of immune response that develops among other leukocytes. The TH1 response produces a cytokine profile that supports inflammation and activates mainly certain T cells and macrophages, whereas the TH2 response activates mainly B cells and immune responses that depend upon antibodies. TC cells are activated when they interact with an antigen-class I MHC complex on the surface of an altered self-cell (e.g., a virus-infected cell or a tumor cell) in the presence of appropriate cytokines. This activation, which results in proliferation, causes the TC cell to differentiate into an effector cell called a cytotoxic T lymphocyte (CTL). In contrast to TH cells, most CTLs secrete few cytokines. Instead, CTLs acquire the ability to recognize and eliminate altered self-cells.
Another subpopulation of T lymphocytes—called T suppressor (TS) cells—has been postulated. It is clear that some T cells help to suppress the humoral and the cell-mediated branches of the immune system, but the actual isolation and cloning of normal TS cells is a matter of controversy and dispute among immunologists. For this reason, it is uncertain whether TS cells do indeed constitute a separate functional subpopulation of T cells. Some immunologists believe that the suppression mediated by T cells observed in some systems is simply the consequence of activities of TH or TC subpopulations whose end results are suppressive.
The natural killer cell was first described in 1976, when it was shown that the body contains a small population of large, granular lymphocytes that display cytotoxic activity against a wide range of tumor cells in the absence of any previous immunization with the tumor. NK cells were subsequently shown to play an important role in host defense both against tumor cells and against cells infected with some, though not all, viruses. These cells, which constitute 5%-10% of lymphocytes in human peripheral blood, do not express the membrane molecules and receptors that distinguish T- and B-cell lineages. Although NK cells do not have T-cell receptors or immunoglobulin incorporated in their plasma membranes, they can recognize potential target cells in two different ways. In some cases, an NK cell employs NK cell receptors to distinguish abnormalities, notably a reduction in the display of class I MHC molecules and the unusual profile of surface antigens displayed by some tumor cells and cells infected by some viruses. Another way in which NK cells recognize potential target cells depends upon the fact that some tumor cells and cells infected by certain viruses display antigens against which the immune system has made an antibody response, so that antitumor or antiviral antibodies are bound to their surfaces. Because NK cells express CD16, a membrane receptor for the carboxyl-terminal end of the IgG molecule, called the Fc region, they can attach to these antibodies and subsequently destroy the targeted cells. This is an example of a process known as antibody-dependent cellmediated cytotoxicity (ADCC). The exact mechanism of NK-cell cytotoxicity, the focus of much current experimental study, is described further in Chapter 14.
Several observations suggest that NK cells play an important role in host defense against tumors. For example, in humans the Chediak-Higashi syndrome—an autosomal recessive disorder—is associated with impairment in neu-trophils, macrophages, and NK cells and an increased incidence of lymphomas. Likewise, mice with an autosomal mutation called beige lack NK cells; these mutants are more susceptible than normal mice to tumor growth following injection with live tumor cells.
There has been growing recognition of a cell type, the NK1-T cell, that has some of the characteristics of both T cells and NK cells. Like T cells, NK1-T cells have T cell receptors (TCRs). Unlike most T cells, the TCRs of NK1-T cells interact with MHC-like molecules called CD1 rather than with class I or class II MHC molecules. Like NK cells, they have variable levels of CD16 and other receptors typical of NK cells, and they can kill cells. A population of triggered NK1-T cells can rapidly secrete large amounts of the cytokines needed to support antibody production by B cells as well as inflammation and the development and expansion of cyto-toxic T cells. Some immunologists view this cell type as a kind of rapid response system that has evolved to provide early help while conventional TH responses are still developing.
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