CHAPTER 14 CELL SURFACE MARKERS OF T-CELLS, B-CELLS AND MACROPHAGES

An understanding of the distinct families of molecules present on different cells of the immune system provides the tools for distinguishing these cell types in both diagnosis and therapy, as well as understanding the molecular basis for many lymphoid cell functions. Diagnostic MARKERS for T-cells include the T-cell receptor (TcR) with its associated CD3 signaling complex as well as CD5 and the E-receptor (CD2). The presence of membrane immunoglobulin (mIg), which functions as antigen receptor, is diagnostic for B-cells. Complement receptors (CR) and Fc receptors (FcR) which can mediate opsonization, and MHC Class II molecules which are important in antigen presentation, are present on B-cells and macrophages (as well as dendritic cells). Two major classes of T-cells are distinguished by the presence of either CD4 (on TH and Treg) or CD8 (on TCcell). TH cells can be further subdivided into TH1,TH2 and TH17, which produce different combinations of cytokines and have distinct physiological roles.

We know that there are at least three major cell types required for immune responses, namely the T-cell, B-cell and macrophage. Conventional microscopy is only of limited usefulness in distinguishing these cells, and is of no use whatsoever in distinguishing various lymphocytes one from another (T-cells from B-cells, or different subpopulations of either). Different classes of cells can readily be distinguished, however, by virtue of the fact that they express unique combinations of molecules in their membranes. Our knowledge of the different cell types involved in immune responses is a direct result of the development of reagents to distinguish these various cells by their cell surface markers. We will see that such markers include molecules distinguished either by antibodies directed against them, or else by their ability to bind various other molecules or cells. SOME MARKERS ON HUMAN T-CELLS, B-CELLS, AND MACROPHAGES MHC Ig TcR(&CD3) FcR CR ClassII __________________________________________ + + + + Table 14-1 + + + +

T-cell B-cell Macrophage

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Ig. One defining characteristic of B-cells is the presence of membrane immunoglobulin, which T-cells lack. These immunoglobulins are IgMS (2L2 IgG-like subunits) and IgD, they are anchored in the cell membrane through a specialized C-terminal domain of the heavy chain, and they function as the cells' antigen-specific receptors. Memory B-cells bear membrane-bound isotypes other than IgM, representing whatever Ig the cell will begin secreting upon antigenic stimulation. TcR. The T-cell antigen receptor (TcR) is the principle defining marker of all T-cells. This molecule is used by the T-cell for specific recognition of MHC-associated peptide antigens, discussed in Chapter 12. Also associated with the TcR is a complex of proteins known as CD3, which participate in the transduction of an intracellular signal following TcR binding to its cognate MHC/antigen complex. FcR. Various cells including B-cells, PMNs and macrophages have molecules on their surface known as Fc-Receptors, which are able of binding IgG through its Fc region. Aggregated or antigen-bound IgG binds much more strongly to these receptors than free, soluble IgG, so that B-cells or macrophages taken directly from an animal will normally have little or no Fc-bound Ig on their surface. The function of Fc-receptors on macrophages is easy to understand in that they mediate opsonization. The function of these receptors on Bcells is less clear, and most likely is involved in regulating humoral responses. When determining if a cell bears surface Ig (to identify B-cells), one must always be careful to account for the possibility of passive Fc-binding (especially to monocytes and/or granulocytes), which may give misleading results. CR. B-cells and macrophages can also bind various complement components, either in free form or as part of an immune complex, by their complement receptors (CR), of which there are at least five known forms. One form of CR known as "CR1" binds C3b and C4b, and is expressed on macrophages, PMNs, B-cells and erythrocytes. As is the case for Fc receptors, the presence of CR on macrophages clearly enhances opsonization (mainly through binding of C3b), while the significance of CR1 on B-cells is less clear. The ability of CR1 to bind C4b is the basis for its role in the normal clearance of soluble immune complexes by erythrocytes (see Chapter 5). T-cells lack complement receptors. MHC Class II. Class II MHC molecules in humans include DP, DQ and DR. (In the mouse they are known as Ia molecules, or I-region antigens). Unlike MHC Class I, Class II molecules are not expressed on all cells; they are present on B-cells, macrophages and other antigen-presenting cells, but not on cells of most non-lymphoid tissues. They are not expressed on "resting" T-cells, but may be induced at low levels when the T-cell is activated. T-cell receptors on "helper"-type (CD4+) T-cells recognize specific antigenic peptides only when the peptides are associated with MHC Class II molecules on the surface of an antigenpresenting cell (discussed in Chapters 12 and 15).

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ANTIBODIES WHICH DISTINGUISH HUMAN T-CELL SUBPOPULATIONS T-cells consist of a variety of subpopulations, each of which can carry out one or more specific immune functions. One important way in which these subpopulations can be distinguished is by the use of the two cell surface markers CD4 and CD8. Distinct Classes of T-Cells Class of Cells TH1 Cell Function Helper cell for CMI, stimulates TC maturation and activity; recruits macrophages in generating DTH response.

TH2

TH

Classical T-helper; stimulates B-cell proliferation and differentiation. T cell with pro-inflammatory role, facilitates activity of other cells, including Tc, NK and macrophages Regulatory T-cell; suppresses development or execution of both B-cell and T-cell immune responses

(CD4+) TH17

Treg

TC

(CD8+)

Cytotoxic T-cell; kills antigen-bearing target cells; acts as effector cell in graft rejection and other cell-mediated immune responses Table 14-2

Mature T-cells express only one or the other of these two cell surface antigens, whose molecular roles in cell cooperation we have discussed in Chapter 12. One class of T-cells is CD4+CD8- and includes the "helper" or TH cells which may be involved in cooperative interactions with either B-cells (TH2) or with other T-cells (TH1), or play other proinflammatory roles (TH17). Treg or regulatory T-cells, which play an important role in tolerance (see Chapter 18), also express this CD4+phenotype. The second major category of T-cells defined by these two markers (CD4-CD8+) encompasses the "cytotoxic" or TC cells which have important effector functions in graft rejection and other cell-mediated immune reactions. The helper class of T-cells can be divided into three sub-categories, based not on cell surface markers but on the lymphokines they produce and the kinds of immune reactions in which they participate. TH2 cells are the classical TH helper cells originally defined by their role in providing B-cell "help" in humoral immune responses. TH1 cells, on the other hand, are 113

"inflammatory" T-cells which provide "help" to other T-cells (e.g. in development of graft rejection) or directly promote inflammatory reactions by their actions on macrophages and PMN's (e.g. in skin DTH reactions). The roles of TH1 and TH2 cells are discussed in more detail in Chapters 12 and15. A more recently defined class of CD4+CD8- TH cell is the TH17 cell, which produces a mix of cytokines including IL-17. This pro-inflammatory cell stimulates the differentiation and activity of a variety of other cells important in protective immune resoponses, including NK cells, PMNs and macrophages. A wide variety of antibodies have been developed which distinguish human T-cell subpopulations. These have been the result of the development of HYBRIDOMA technology (see APPENDIX 13), and a few of the commercially available antibodies are listed below:

"CD" Name CD2 CD3* CD4* CD5 CD8*

Anti-human monoclonal Antibodies (OKT-11, Leu-5) (OKT-3, Leu-4) (OKT-4, Leu-3) (OKT-1, Leu-1) (OKT-8, Leu-2)

Distribution on Human Cells All T ("E-receptor") All T TH ,Treg (+ macrophage) All T TC

*You should be familiar with the distribution of these three markers and their biological significance. Table 14-3

The CD4 and CD8 molecules (and the antibodies directed against them) are of particular importance in clinical medicine. The loss of CD4-bearing T-cells, for example, is associated with disease progression in AIDS, and determination of the CD4:CD8 ratio is a standard tool in the evaluation of HIV-positive patients. CD4 itself also plays an important role in HIV infection, as it is the major receptor by which the virus enters those cells which it infects (Tcells and macrophages). The monoclonal antibodies to human lymphocyte antigens outlined above have been tremendously useful in diagnostic medicine for evaluating the immune status of patients. Use of these reagents can yield information about the relative abundance of different functional Tcell subsets, which is much more useful than knowing only the total number of lymphocytes, or even the relative number of T-cells and B-cells. In addition, such reagents can be used therapeutically for specific removal of a particular T-cell subset, as, for example, the use of anti-CD4 to inhibit TH cell function in autoimmune disease. 114

A note on nomenclature: The confusing naming systems historically associated with many cell surface markers in different species over the years has been made more rational by the adoption of a standard nomenclature system. If cell surface antigens in different species (which often are assigned unrelated names as they are discovered) are determined to be homologous by molecular characterization, they are assigned a standard name in the CD series ("Complex of Differentiation"). Thus, CD3 molecules in human, mouse, rat, etc all have similar sequences and structures. Despite such structural homology, however, identical CD antigens do not necessarily have the same tissue distribution or function in different species. For example, CD5 in humans is present on all T-cells, whereas CD5 in mice (originally called Ly-1) is present only on the helper subpopulation of T-cells. This is just one example of many differences in the patterns of differentiation of T-cells between humans and other mammalian species. FUNCTIONAL MARKERS FOR LYMPHOCYTE SUBSETS: MITOGENS AND OTHERS While the presence of one or another cell surface antigen has long been the most widely used method for distinguishing different lymphoid cells, other approaches have also been used, for example the use of mitogens to activate different classes of lymphocytes. A mitogen is any substance which stimulates a non-cycling cell to undergo mitosis, and many mitogens are members of a large group of carbohydrate-binding molecules known as lectins. Concanavalin A (ConA) is one such lectin, which selectively stimulates T-cells to divide in both humans and rodents; the proliferative response of a lymphocyte population to ConA can therefore be used as a crude measure of the proportion of T-cells it contains. Another lectin, Pokeweed Mitogen (PWM), is selective for rodent B-cells and can therefore be used experimentally as a measure of the proportion of B-cells in a population. Other functional characteristics can also be used to distinguish various populations of lymphocytes. The ability to transfer a DTH response, for instance, is the defining characteristic of TD (or TDTH) cells (which are mostly TH1), and can be used to detect their presence. As another example, macrophages/monocytes (and granulocytes) can be readily distinguished from lymphocytes by virtue of their ability to adhere tightly to glass or plastic surfaces, as well as by their relative resistance to the toxic effects of ionizing radiation. For example, adherence can be used to selectively remove monocytes from a mixed population of blood leukocytes, leaving behind mostly lymphocytes. Exposing blood leukocytes to sufficient doses of ionizing radiation, on the other hand, will selectively kill lymphocytes, and the surviving cells will be mostly monocytes and granulocytes.

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CHAPTER 14, STUDY QUESTIONS: 1. 2. 3. Describe those cell surface markers which characterize and distinguish B-CELLS, TCELLS AND MACROPHAGES. What functional roles do FcR and CR play in the immune system? Describe the distinct roles of TH1 and TH2 cells in immune responses. Which cell markers do they share? Which markers may be used to distinguish them?

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