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Lymphocytes

University of Manitoba, Manitoba Cancer Treatment and Research Foundation, Manitoba, Canada

	Abstract

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Mechanisms in Hematology is a book with an accompanying interactive CD-ROM designed to assemble basic concepts that underlie clinical understanding and progress. It is presented as a concise text with a series of diagrams that distill diffuse information into a compact form. The interactive CD, in particular, brings many of the processes "to life" as details of the more complex pathways are conveyed in clear visual images. The text begins with the basic molecular biology that underlies hematological and oncological physiology/pathology––cell signaling, adhesion molecules and apoptosis. This is followed by sections, among others, on hematopoiesis, iron, B₁₂ and folate metabolism, neutrophil function, immunoproteins, chemotherapy and coagulation. With the permission of the authors and publisher, Stem Cells has reproduced the section on lymphocytes, which we think our readers will enjoy.

	Introduction

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"To be or not to be—that is the question." Hamlet, Act III Scene I.

On the peripheral blood smear, the lymphocyte appears the least interesting of all the leukocytes—the monotonous sameness of appearance gives no clue to its complex history, its present function or its future; nor can we differentiate between the T cells responsible for cellular immunity and the B cells that provide humoral immunity. In the peripheral blood about 70% of circulating lymphocytes are T cells, 15% are B cells and 15% are natural killer (NK) cells.

The lymphoid immune system consists primarily of quiescent T and B cells—each potentially responsive to a unique antigen. Following recognition of foreign antigen, the immune attack is mediated by expanded clones of antigen-specific T and B lymphocytes. NK cells exist as preactivated cytotoxic cells: their effector response is rapid, without the need for prior activation.

	B Lymphocytes

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The function of the B lymphocyte is the production of antibody in response to external antigens. Plate 1 traces the history of B cell development from stem cell to differentiated plasma cell and depicts the cytokine stimuli, the gene rearrangements required for immunoglobulin synthesis and the cell surface markers, referred to as CDs (clusters of differentiation), that characterize each evolutionary stage. B cells are derived from bone marrow CD34⁺ stem cells. Under the guidance of stromal cells and the stimulus of IL-7, but in the absence of antigen, they acquire the potential (the repertoire) for antibody production. Following the development of this potential, they leave the marrow for the peripheral lymphoid organs (lymph nodes, spleen, gut) where antigen stimulation drives their proliferation and maturation to mature B cells and plasma cells producing specific antibodies.

Bone Marrow Phase of Differentiation
This phase of B cell development is antigen-independent as the cells acquire the capacity to respond to an unlimited number of antigens; each individual cell, however, will express only a single specific antibody. This stage is characterized by the synthesis of monospecific immunoglobulin (Ig) molecules that will serve both as the cell surface antigen receptor and, on release, as the unique antibody to the same antigen. Somatic rearrangements and selection of the gene segments for Ig heavy (H) and light (L) chains provide the broad repertoire of gene combinations that yields the spectrum of antibodies required to combine with the countless antigens in the environment.

In the marrow, lymphocyte development depends on the productive and initially random rearrangement of H chain and L chain genes to generate IgM (Plate 2). The H chain variable domains are encoded by three separate gene segments on chromosome 14, designated V_H, D_H and J_H. The rearrangement necessary to produce H chains occurs in two steps. In the early pro-B cell, H chain gene rearrangement begins with selection of the diversity (D_H) and joining (J_H) segments to produce the D_HJ_H complex; in the pre-B cell, the variable (V_H) region is selectively rearranged and joins D_H J_H to form the V_H D_H J_H gene. Finally, in conjunction with the Cµ gene of the constant (C) region, this rearranged gene is transcribed and processed as the IgM heavy chain (V_H D_H J_H — Cµ) that is expressed in the cytoplasm. With the appearance of H chains in the cytoplasm, light chain rearrangement begins on chromosome 2 encoding the kappa () chain gene. If this step is successful, light chains join the heavy chains to produce a complete IgM molecule. If chain rearrangement fails, the cell initiates rearrangement of the lambda () light chain locus on chromosome 22; if successful, a functional light chain will result in the formation of IgM. Successful rearrangements of the H and L chain genes occur in only about 20% of cells. Failure to achieve functional gene rearrangement results in disappearance of the cell by apoptosis—("To be or not to be!"). During this random rearrangement process, some immature B cells in the marrow develop receptors that recognize self-antigen; to prevent the production of antibodies against host tissues, cells that exhibit autoreactivity also undergo apoptosis with resultant clonal deletion.

On gene transcription and translation, complete IgM appears in the cytoplasm followed by its expression on the cell surface as a receptor capable of binding antigen. This marks the end of the first stage of differentiation—the cells now migrate to the peripheral lymphoid organs.

Peripheral Phase of Differentiation
The next stage of maturation takes place in the spleen, lymph nodes, and other lymphoid-bearing tissues. This stage of differentiation is antigen-dependent; it is driven by foreign antigen and by cytokines (the interleukins). Within the follicles and germinal centres of the peripheral lymphoid system, the B cells interact with T cells and follicular dendritic cells. The most important regulators of B cell differentiation are helper T (CD4) cells scattered throughout the lymphoid follicles. This "help" is delivered by cell-cell interaction and by the cytokines produced by these CD4 cells. In the periphery, there are B cells that have never encountered antigen (naive B cells), others that carry the message of previous encounters (memory cells). In both these groups, designated as resting B cells, Ig is present on the cell membrane but little if any is secreted.

Antigen binding to naive B cells is often insufficient to stimulate antibody production—additional signals are required from helper T cells. When B cell receptors bind antigen, the antigen is internalized and processed to small peptides; the peptides are returned to the cell surface bound to a specialized transport protein termed the major histocompatibility complex (MHC) molecule. (These proteins were first recognized by their participation in the immune response to transplanted tissues—their name refers to this function). MHC molecules are of two types and serve two different pathways. MHC class II molecules present peptides, derived from proteins of extracellular origin, processed in the endosomes of B cells and macrophages. This peptide-MHC class II complex appears on the surface of the cell and co-operates selectively with T cells bearing the CD4 ligand. MHC class I molecules carry the peptides derived from foreign (virus) proteins, processed within the cytosol, to the membrane where the MHC class I molecule/peptide complex is recognized by T cells bearing the CD8 ligand.

The MHC class II/peptide membrane complex is recognized by the antigen-specific CD4 helper T cells that now activate B cells and stimulate clonal expansion by inducing B cells to proliferate and their progeny to differentiate into antibody-secreting plasma cells. The early antibody response is marked by the appearance of IgM antibodies in the plasma; IgG and IgA antibodies, responsible for the major and prolonged biological response, appear later. As B cells interact with antigen, helper T cells induce DNA rearrangements that result in the formation of new classes of antibody through isotype switching. The V_H D_H J_H region of the heavy chain gene now combines with C_H regions other than µ (, , , ) to produce classes of Ig other than the original IgM, i.e. IgG, IgA, IgD, or IgE; this is designated "class switch." Class switch is dependent on the binding of the B cell receptor CD40 to the CD40 ligand on activated T cells. In congenital absence of the T cell CD40 ligand, isotype switch does not occur—IgG and IgA are not produced. Class switch provides the IgG antibodies that can move outside the intravascular space and also cross the placenta, and IgA that can be transported to mucosal surfaces.

There is a continuous recirculation of B cells through lymphoid follicles, peripheral blood and lymph. Millions of B cells are formed daily in the marrow; the auto-reactive cells are removed at that site. Those B cells that emerge may become: (1) short-lived naive cells that do not encounter antigen, and soon undergo apoptosis, (2) those stimulated by antigen that become long-lived memory cells, and (3) those that evolve into Ig-secreting plasma cells.

Plasma cell transformation is marked by characteristic morphological changes including cell enlargement, a cartwheel arrangement of chromatin blocks in the nucleus, and the development of a highly organized endoplasmic reticulum. The Ig synthesized in these cells packs the endoplasmic reticulum, is then channeled to the Golgi apparatus where glycosylation occurs and, finally, is transported to the surface for secretion.

	T Lymphocytes

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T cells also arise from bone marrow stem cells, then migrate to the thymus where in the course of maturation their specific character and subsequent role are determined. In the infant and child there is rapid proliferation of T cells within the thymus—this key role the thymus plays in generating T lymphocytes declines at puberty as the thymus undergoes substantial involution. The thymus is essentially autonomous: the stimulus for T cell proliferation is dependent exclusively on signals arising within the thymic stroma. As a result of screening for tolerance to self-antigens, over 90% of generated T cells undergo apoptosis within the thymus. The function of the T cell is defined by its membrane receptor. The repertoire of the T-cell receptors (TCRs) is dictated by the random somatic rearrangement of gene segments—analogous to Ig synthesis in the B cell. Sequential V, J and C gene rearrangement is followed by expression of the TCR on the surface membrane. Each TCR is specific for a single antigen; the large number of combinations, made possible by gene rearrangement, provides the vast receptor diversity.

The role of B cells is humoral immunity—the production of antibody to extracellular antigens; the role of T cells is to combat intracellular pathogens and destroy abnormal cells by a cell-mediated immune response. Depending upon the nature of the target cell or the type of infecting organism, cell-mediated immunity is accomplished by: (1) direct cell kill—cytotoxic CD8 T cells, (2) activating an inflammatory response within the infected cell—inflammatory CD4 T cells, also called Th1, or (3) stimulating antibody production by co-operating B cells—helper CD4 T cells (Th2).

T cells do not respond to large antigen molecules: they require prior antigen processing by the antigen presenting cells (APCs) resident in lymphoid tissue. The antigen is internalized and degraded to small peptides by the APC, then transported to the cell surface by the MHC molecules. The displayed MHC/peptide complex interacts with and activates the T cell carrying the appropriate matching TCR.

There are three types of APCs: (1) Dendritic cells process and present proteins of viruses and bacteria that have gained entry into these cells and multiply in the cytosol—these peptides are primarily presented on the surface of the cell in association with MHC class I molecules; some viral fragments are processed in endosomes and these are displayed by MHC class II molecules. (2) Macrophages engulf microorganisms, degrade them within cellular vesicles, and present the peptides on MHC class II molecules. (3) B cells internalize and degrade the antigen bound to its surface immunoglobulin; the peptides are processed within endosomes, then displayed by MHC class II molecules. The continuous re-circulation of T cells through nodes, spleen, lymph and blood brings naive T cells into contact with the APCs.

Within chronic inflammatory sites the lymphocytes interact with the endothelium by mechanisms similar to that of neutrophil recruitment. The surface displayed L-selectin brings the rolling lymphocyte into close contact with the endothelium where the endothelial expressed P- and E-selectins mediate tighter binding. Subsequent lymphocyte expression of the integrin CD11a/CD18 and its interaction with the endothelial cell intercellular adhesion molecules (lCAM-1) produce surface spreading of the cell prior to its migration through the inter-endothelial junctions.

A naive T cell may circulate for many years without differentiating, but when this cell encounters its specific antigen presented by an APC, it is primed to proliferate and its progeny to differentiate into effector T cells. The effector T cells leave the lymphoid tissue, circulate in the bloodstream and migrate to sites displaying foreign antigen. Cytotoxic T cells recognize MHC class I/peptide complexes. Inflammatory and helper T cells interact with MHC class II/peptide complexes. The interaction of a TCR with the MHC/peptide complex requires the participation of a co-receptor: CD8 for the cytotoxic T cell, and CD4 for the inflammatory and helper T cells. These T cells are often referred to as CD8⁺ and CD4⁺ cells, respectively. In the peripheral blood, approximately 40% of the total lymphocyte population are CD4⁺ cells and 30% are CD8⁺.

T Cell Response
T cell and NK cell immunity represent complementary arms of the cellular immune response; they have a pivotal role in protective immunity. The T cell response is determined by the type of effector T cell and the nature of the target cell (Plate 3).

(1) CD4⁺ Cells   Proliferating CD4⁺ cells differentiate into two types of effector cells: the inflammatory T cell (Th1) activates macrophages, and the helper T cell (Th2) activates B cells.

A) In response to MHC class II/peptides displayed on infected macrophages, the Th1 T cell, through its TCR in concert with its CD4 co-receptor, binds to and activates the macrophage. The T cell binding and release of IL-2 mediates the kill of the intracellular invaders or the destruction of foreign antigen. The effector T cell may then disengage and move to a second similar target or be stimulated to clonal expansion.

B) The Th2 helper T cells home to B cells bearing specific processed antigen on MHC class II molecules; the TCR, with its CD4 co-receptor, binds to the B cell. Secondary binding of B cell CD40 to the CD40 ligand on the T cell, together with T cell cytokine release, stimulates the B cell to proliferation and antibody synthesis. The close contact of T and B cells is largely mediated by the CD40 ligand-CD40 interaction. B cell activation provides a reciprocal stimulus for T cell activation by sustaining the ligand expression and cytokine release. These interactions take place as the B cells traverse the T cell-rich areas in lymph nodes and spleen.

(2) CD8⁺ Cells   The cytotoxic CD8⁺ T cell (CTL) is particulary effective against cells that have become infected by an intracellular pathogen (virus or bacterium) or that have been altered by malignancy. Such cells are recognized as "foreign" by the peptides displayed on MHC class I molecules. The CTL cell binds via its specific TCR; this docking is augmented and stabilized by the binding of the CD8 surface molecule. The binding of the two ligands to the target cell signals T cell activation. This antigen-specific directional killing may be mediated by either: (A) CTL release of perforins and granzymes; the former producing a membrane spanning pore allowing entry of the granzyme protease that triggers DNA fragmentation, or (B) a second mechanism that depends on the appearance of Fas ligand on the activated T cell; it binds to the Fas antigen expressed on the target cell and activates apoptosis (Plate 3).

NK Cells
These cytotoxic effector lymphocytes are a separate subset of T cells. Morphologically they resemble the other lymphocytes but for the large azurophilic granules in their cytoplasm. They too are cytotoxic, but unlike CD8 cells, they recognize only those target cells that do not exhibit normal expression of MHC class I molecules—a property exhibited by some tumor cells and some virus-infected cells. Thus their role is complementary to the cytotoxic CD8 T cell which requires MHC class I/peptide display. T cells and NK cells arise from a common progenitor and express several cell surface molecules in common. However, NK cells do not express antigen-specific cell surface receptors. NK cells recognize only those cells that express a down-regulated, altered or absent MHC class I molecule ("the missing-self" hypothesis). The rapid production of -interferon by NK cells takes place before the clonal expansion and differentiation of antigen-specific T cells. Thus, NK cells have a particular importance in the earliest stages of pathogen invasion where they respond both by cytoxicity and by cytokine production. Cytotoxicity is mediated by the same mechanisms that operate in other CTLs.

	Clinical Correlates

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Immunodeficiency
Congenital or acquired immunodeficiency is present when components of the immune system are absent or defective. The result is susceptibility to various infections predicated on the nature of the immunological defect.

Congenital immunodeficiency diseases include primary defects in B cell or T cell development, and primary T cell defects with secondary B cell functional failure. These can be divided into: (1) those with, predominantly, defects in antibody production, and (2) those with both humoral and cellular compromised immunity. Examples of these are:

1. Failure of antibody synthesis.
   
   A) Pure B cell deficiency (Bruton's X-linked agammaglobulinemia) in which a defective btk gene, coding for a tyrosine kinase, leads to a defect in immunoglobulin gene rearrangement with maturation arrest at the level of the pre-B cell.
   
   B) The X-linked hyper-IgM syndrome is a T cell defect of the CD40 ligand. Although B cell expression of IgM is normal, in the absence of stimulation of B cell CD40 through the T cell CD40 ligand, the B cells fail to undergo class switch. These patients have high levels of IgM but IgG and IgA are not synthesized.
   
   
   
   
2. Severe combined immunodeficiency disease (SCID).
   
   A) An X-linked defect in the T cell IL-2 receptor chain: there is loss of primary T cell activation and secondary B cell stimulation.
   
   B) Congenital adenosine deaminase (ADA) deficiency. This results in high levels of non-metabolized adenosine and deoxyadenosine derived from DNA catabolism during growth. Deoxyadenosine is particulary toxic to thymocytes in the developing fetus.
   
   C) Congenital thymic aplasia—absence of the site of T cell maturation (DiGeorge Syndrome).
   
   D) MHC class II deficiency (the "bare lymphocyte syndrome") in which there is a defect in the transcription of the MHC II gene. It is characterized by the failure of expression of MHC II surface molecules on bone marrow-derived APCs.
   
   
   
   

B Cell Malignancies
Acute Lymphoblastic Leukemia (ALL) is a proliferative disease of either pro-B or pre-B lymphoid cells. The pro-B group is characterized by arrest at the stage of the early gene rearrangement of the D_H and J_H segments of H chain. In pre-B ALL, there is complete heavy chain gene rearrangement of V_H D_H J_H Cµ with the production of cytoplasmic but not membrane-associated IgM. Most of these cells display the surface markers CD34 (a stem cell adhesion molecule) and CD10 (a metalloproteinase) known as CALLA—the common acute lymphocytic leukemia antigen (Plate 1).

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of long-lived B cells that carry the T cell marker CD5 normally present on only a small number of B cells. Other surface markers include CD20—a transmembrane protein that regulates B cell activation, CD21—a receptor for the C3b component of complement, CD22—an adhesion molecule, and CD23—a C-type lectin. That CD5 B cells can form antibodies against self-antigens may explain the high incidence of autoimmune hemolytic anemia and thrombocytopenia in CLL. Because CLL lymphocytes have increased levels of Bcl-2 they are resistant to apoptosis: this may be the primary mechanism for the accumulation of long-lived lymphocytes that flood the bone marrow, spleen, nodes and peripheral blood in this disease.

Burkitt's Lymphoma. This B cell neoplasm exists in two forms: an endemic African form and a sporadic non-endemic American variety. The Epstein-Barr virus (EBV) is associated with 95% of the endemic form and 15% of the non-endemic form. The CD21 marker (receptor for complement C3b) on these cells binds EBV—a potent stimulus for B cell proliferation.

The chromosomal rearrangements in Burkitt's lymphoma and in follicular lymphoma are a study in similarity and contrast (Plate 4). In Burkitt's lymphoma there is a characteristic t(8:14) reciprocal translocation in which the c-myc oncogene of chromosome 8 is translocated into the IgH-chain locus on chromosome 14. The c-myc oncogene dictates a transcription factor that upregulates gene expression and increases cell proliferation. The breakpoint on chromosome 14 is within the D or J regions of the H chain in endemic Burkitt's and within the µ region in the sporadic form. This brings c-myc under the control of the immunoglobulin gene enhancer region leading to high levels of expression that support rapid cell proliferation.

Follicular Lymphoma. In 85% of cases of this low-grade lymphoma there is a reciprocal translocation between chromosomes 14 and 18—t(14:18). This translocation moves the oncogene bcl-2 from chromosome 18 into juxtaposition with the enhancer region of the IgH-chain gene on chromosome 14. The resultant over-expression of bcl-2 confers a survival advantage on these B cells which now defy apoptosis. The continuing accumulation of these cells, rather than their rapid proliferation, is a hallmark of this low-grade lymphoma. In some patients, a subsequent genetic event involving a gene for cell proliferation (e.g., ras) may transform indolent disease into a more aggressive lymphoma.

Mantle Cell Lymphoma. This is another example of a lymphoma associated with a gene translocation into the enhancer region of the Ig heavy-chain gene on chromosome 14. It is an intermediate-grade lymphoma associated with gastrointestinal infiltrates and Waldeyer's ring involvement. In this lymphoma, a t(11:14) translocation brings the gene for cyclin D on chromosome 11 into juxtaposition with the H-chain gene enhancer. The overexpression of cyclin D, a regulator of the G₁ phase of the cell cycle, results in cell cycle deregulation.

T Cell Malignancies
These are far less common than B cell malignancies. Depending on the stage of cell evolution they fall into two main categories: (1) evolution from pre-thymic and early-thymic stages gives rise to acute lymphoblastic T cell leukemia or lymphoma, and (2) those arising from post-thymic cells are the T-cell lymphomas and 5%-10% of cases of chronic lymphocytic leukemia. Most primary cutaneous lymphomas, including mycosis fungoides and the Sezary Syndrome, are T cell-derived.

Reproduced courtesy of Core Health Services Inc., Concord, Ontario, Canada.

View larger version (39K): [in this window] [in a new window]   Plate 1. B cell maturation.

View larger version (22K): [in this window] [in a new window]   Plate 2. The synthesis of IgM monomer.

View larger version (31K): [in this window] [in a new window]   Plate 3. Effector T cells.

View larger version (26K): [in this window] [in a new window]   Plate 4. Chromosomal translocations—Burkitt's lymphoma—follicular lymphoma.

	References

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