Figure 73–6 summarizes the expression of antigens on cells of the B-lymphocyte lineage, including committed progenitor B cells and pre-B cells. Chapter 74 discusses these cells and the maturation stages they represent. Figure 73–6 also lists antigens that are expressed or increased upon B-cell activation. Of the B-cell–associated antigens that are commonly used, only a few are restricted to cells of the B lineage. Of these antigens, only CD20, CD22, and Pax 5 are not found on other cell types. Pax 5, a transcription factor, is a “master regulator” of B-cell development32,33 that is expressed from the precursor stage through all B-cell maturation until it is lost at the plasma cell stage. Demonstration of monoclonal surface Ig allows diagnosis of clonal, neoplastic B cells. CD20 is the target of rituximab, a monoclonal antibody commonly used for treatment of B-cell neoplasms. CD19 is restricted mostly to B cells, but may be expressed weakly by follicular dendritic cells. CD19 is expressed by B cells at all stages of maturation, including the committed B-cell progenitor and most normal plasma cells. As such, it is the best-defined pan–B-cell surface antigen.
Clinically useful antigens expressed during B-lymphocyte maturation. The intensity of the antigen expression at each stage of B-lymphocyte maturation is depicted by gradient density of bars on the graph. c, cytoplasmic; s, surface.
In addition to the CD antigens and Igs, B cells express the three major histocompatibility complex (MHC) class II antigens: DR, DP, DQ. These antigens are heterodimers of heavy chains and light chains that are encoded by genes within the D complex of the human leukocyte antigen (HLA) complex (Chap. 137). MHC class I antigens are expressed on all nucleated cells.
B-1 B Cells and CD5+ B Cells
B-1 B cells have distinctive activation requirements and high levels of CD44 and interleukin (IL)-5 receptor α (IL-5Rα). They proliferate more rapidly than other B cells to stimuli such as IgM crosslinking, possibly because of having constitutively activated nuclear signal transducer and activator of transcription 3 (STAT3).34 Many, but not all, B-1 cells express CD5, a 67-kDa transmembrane glycoprotein that is more brightly expressed by T cells. These cells are designated CD5 B cells.35 B-1 B cells do not express other T-cell markers but do express all other pan–B-cell surface antigens. Various agents modulate B-cell expression of CD5.36 B-1 B cells are found in umbilical cord blood,37 adult blood, the pleura and peritoneum, and all major secondary lymphoid organs; they are rare in the marrow.38 These cells apparently are enriched for cells that spontaneously produce polyreactive autoantibodies.39,40,41
Many B cell differentiation antigens are not expressed by the mature plasma cell, including CD20, Pax-5, surface Ig, and HLA class II antigens (see Fig. 73–6). Of the cells of the B lineage, plasma cells are distinctive in that they express CD138 and bright CD38.42 Clonal plasma cell neoplasms usually have antigen expression distinct from normal plasma cells including aberrant expression of CD20, CD28, CD56, and CD117. Clonal plasma cells usually aberrantly lack expression of CD19 and CD27.43
T-LYMPHOCYTE AND NATURAL KILLER CELL ANTIGENS
Figure 73–7 and Table 73–1 summarize the expression of antigens on cells of the T-lymphocyte and NK lineages. All lymphocyte progenitors originate in the marrow, but T lymphocytes have their own special organ for maturation—the thymus—whereas NK cells appear to differentiate in secondary lymphoid tissue (Chap. 6).
Table 73–1.Mature Natural Killer and T-Lymphocyte Subsets ||Download (.pdf) Table 73–1. Mature Natural Killer and T-Lymphocyte Subsets
|NK or T-Cell Subset ||Antigens |
|CD4+ helper T cells || |
CD2, CD3, CD4, CD5, CD7, and TCR α/β
Subsets selectively express CD10, CD25,49 CD57,99 and FoxP3100
|CD8+ cytolytic T cells || |
CD2, CD3, CD5, CD7, CD8, and TCR α/β
Subsets selectively express CD16,55 CD56, CD57, cytolytic enzymes101
|NK cells || |
CD2, CD7, KIRs (multiple), NKp46 (NCR1)
Negative for CD3, TCR (α/β or γ/δ)
Subsets express either CD16-negative, dim and CD56 bright, or CD16 bright and CD56 dim54
|γ/δ T cells || |
CD2, CD3, CD7, TCR γ/δ
Usually negative for CD4
Subsets express CD5, CD8, and cytolytic enzymes
Clinically useful antigens expressed during T-lymphocyte maturation. The intensity of the antigen expression at each stage of T-lymphocyte maturation is depicted by gradient density of bars on the graph.
The thymus promotes the development of antigen-specific T lymphocytes and eliminates self-reactive T lymphocytes. There are three general stages of thymocyte maturation based on the surface CD4 and CD8 expression: double negative, double positive, and single positive. These stages have corresponding anatomic localization within the thymus with the least-mature double-negative cells located in the subcapsular area and the most-mature single-positive cells located in the medulla (Chap. 6). The most immature T lymphocytes in the thymus populate the subcapsular areas and express CD2, CD5, and CD7, antigens present on T lymphocytes of all stages. Capsular, “double-negative” thymocytes also express CD1a, cytoplasmic CD3, and terminal deoxynucleotidyl transferase (TdT). The majority of thymocytes are at the “double-positive” stage within the cortical area. These are the cells undergoing positive selection. Once the thymocytes achieve their “education” without dying, they mature to the single-positive stage in the medulla. These varying stages of immature T-cell maturation in the thymus have corresponding cell phenotypes in T-lymphoblastic leukemia/lymphoma.
Small, mature T lymphocytes are the most common lymphocytes in blood. T lymphocytes recognize antigen in the context of the MHC through binding with the T-cell receptor (TCR). Signaling from the TCR involves many membrane proteins, including CD3, a three-subunit complex expressed by early thymocytes and mature T cells.44 It is tightly linked to the T-cell antigen receptor (Chap. 76). Most T lymphocytes have the α/β TCR on their surface, but a few percent of blood lymphocytes have the γ/δ TCR.
CD4 and CD8 Lymphocyte Subsets
Mature T cells express either CD4 or CD8, but not both. CD4, a member of the Ig supergene family, is a single-chain transmembrane glycoprotein.45 CD8 is a 34-kDa dimeric transmembrane glycoprotein.46 Most T cells express the α and β subunits of CD8. CD4 and CD8 act as coreceptors during T-cell activation by antigen. CD4 recognizes MHC II and CD8 recognizes MHC class I (Chap. 76). CD4 also is a coreceptor for the human immunodeficiency virus47 (Chap. 81), as are CCR5 (chemokine receptor 5) and CXCR4 (chemokine-related receptor). The majority of CD8 cells are cytolytic when appropriately activated.
Subsets of CD4+ T cells have helper function for activation and maturation of cytolytic cells or B cells. Other CD4 subsets have regulatory activity including the T-regulatory (TREG) cells that induce immune tolerance, and follicular T-helper (TFH) cells that promote B cell maturation and differentiation in germinal centers. TREG and TFH cells have unique phenotypes. TREG cells express the low affinity receptor for IL-2 (CD25); and the transcription factor forkhead box P3 (FoxP3).48 Follicular helper T-TFH cells express CD10 and CD57. Malignant counterparts to both subsets occur. Adult T-cell leukemia/lymphoma expresses both CD25 and FoxP3 and is associated with marked immunosuppression.49 Angioimmunoblastic T-cell lymphoma has characteristic clonal T cells that express CD10 and CD57 just like TFH cells, and this lymphoma is associated with polyclonal hypergammaglobulinemia and the expansion and proliferation of both B cells and CD21+ follicular dendritic cells.50 T-helper-17 (Th17) cells that secrete IL-17 exhibit critical roles in mucosal defense and in autoimmune disease pathogenesis.51
The NK cell is defined as an effector cell that is not MHC restricted and has the capacity for spontaneous cytotoxicity toward various target cells (Chap. 77). Most NK cells have LGL morphology (see Fig. 73–1E).52 However, not all NK cells have LGL morphology, and not all LGL cells are NK cells. Many are cytolytic T lymphocytes. Cytolytic T lymphocytes and NK cells share many granule contents that can be detected by immunohistochemistry or flow cytometry. These include TIA-1, an RNA binding protein, and several granzymes, which are granule enzymes with serine protease activity.
Despite their relative morphologic homogeneity, NK cells comprise several subpopulations with distinct phenotypes. Human NK cells characteristically express CD16 (FcγRIII) and CD56 but not TCRα/β or TCRγ/δ, CD3, or CD4.53,54 CD8 is found on approximately 30 to 50 percent of NK cells. CD8 on NK cells is dim by flow cytometry and is of the β-homodimer form. CD16 (FcγRIII) is a low-affinity receptor that binds to IgG, which is bound specifically to antigens present on cells targeted for destruction in antibody-dependent cell-mediated cytotoxicity.55 CD16 is expressed on all NK cells, neutrophils, and tissue macrophages. CD56 is the neural cell adhesion molecule and is seen on most NK cells in either low (“dim”) or high (“bright”) density.53,56 This 200-kDa protein is expressed at higher levels following activation.
LYMPHOCYTE SURFACE ANTIGENS
Lymphocyte subsets generally cannot be distinguished from one another by morphology. Most resting lymphocytes appear as small round cells with a dense nucleus and little cytoplasm. However, this homogeneous appearance is deceptive, as these cells comprise many functionally distinct subpopulations.
These subsets can be distinguished through the differential expression of cell-surface proteins, each of which can be recognized by a specific monoclonal antibody. Coupled with the biochemical analyses of the surface molecules that are recognized by each of these antibodies, many lymphocyte surface antigens have been defined.
Typically, it is necessary to monitor for coexpression of two or more cell-surface proteins to define a functional subset of lymphocytes. The same cell-surface protein is often expressed by more than one cell subset. For example, both helper and cytotoxic T cells express CD3, the proteins associated with the TCR for antigen (Chap. 76). Expression of both CD3 and CD4 helps to distinguish mature Th cells from cytotoxic T cells that express CD3 and CD8, and from other cells, such as dendritic cells, that express CD4 but lack expression of CD3 (Chap. 76). As noted above (see “CD4 and CD8 Lymphocyte Subsets”), TREG cells are defined by the coexpression of CD3, CD4, CD25, and cytoplasmic FoxP3.48 For these and other types of lymphocytes, it is the expression of a characteristic constellation of surface and cytoplasmic molecules, rather than the expression of any one particular marker, that generally helps to distinguish one subset of lymphocytes from another.
Fluorescent probes also can be used to identify antigen-specific lymphocytes.57 Each clone of B lymphocytes expresses Ig capable of binding a particular antigen (Chap. 75). The frequencies of B cells specific for one antigen are estimated to range from 1 in 100,000 to 1 in 1,000,000 cells or less. Populations of lymphocytes enriched for B cells binding to a specific antigen can be stained using antigen coupled to probes, allowing for the detection and isolation of antigen-specific B cells using flow cytometry.58 Alternatively, flow-based techniques can be used to monitor for antigen-specific B cells that are activated by contact with antigen.59 T lymphocytes, however, generally recognize antigen in the form of peptides nestled into molecules of the MHC (Chap. 76). Thus identification and isolation of antigen-specific T cells require more complex probes using multimeric complexes comprised of specific peptide antigen complexed with the relevant MHC molecule.57