Immune Effector Cells Lymphocytes

Two pools of lymphocytes are found in the body. The largest pool resides in the secondary lymphoid organs such as the spleen and lymph nodes. Lymphocytes in these organs are usually undergoing stimulation and differentiation. Resting lymphocytes in the peripheral blood represent the smallest pool. At any point in time, only 2% of the total lymphocyte pool circulates in the peripheral blood (Anonymous, 1995a). The number of lymphocytes in the peripheral blood varies with the species. In humans, lymphocytes comprise 20-40%of the total white blood cell population. In rats, over 70% of the total white blood cells are lymphocytes.

Using light microscopy, all lymphocytes have the same morphology and staining characteristics. However, functional studies reveal that there are many lymphocyte subpopulations. Lymphocytes are divided into T and B cells depending on whether the cells are induced to maturation in the thymus (T cells) or the bone marrow or organs analogous to the bursa of Fabricius (B cells) in chickens. From a disease perspective, T cells are involved in inflammatory responses to intracellular bacteria, immunoregulation, and the lysis of tumor cells. Conversely, B cells produce protein antibodies directed toward bacteria and other extracellular pathogens. With the advent of flow cytometric analyses and fluorescence-labeled monoclonal antibodies reacting with cluster of differentiation (CD) markers on lymphocytes, it is possible to further dissect subpopulations of T and B cells. Pan T and B cell markers have been identified. In humans and rodents, all T cells express CD3 markers and all B cells express CD19-CD21 surface proteins. Using antibodies with broad-spectrum CD reactivity, subpopulations of T cells can be identified. T helper/amplifier and T cytotoxic/suppressor cells are identified by the presence of CD4 and CD8 markers, respectively. Based on experimental data, it has become clear that the CD4 and CD8 populations are heterogeneous with respect to immunological function. The CD4 population contains helper cells for antibody production (Th) and amplifier cells (Ta). Subpopulations of CD8 cells include two killer cell subsets (Tel and Tc2) involved in tumor lysis and cells with possible suppressive activity (Ts). The CD4 helper/amplifier populations can be further dissected by defining the presence or absence ofleukocyte common marker isoforms and the CD30 marker. T helper cells that assist B cells in the production of antibodies express CD4, CD45RA+, CD30+ markers. In contrast, CD4 cells that amplify the response (amplifier T cells) express CD4, CD45RO+, CD30-. An alternate nomenclature has been used to divide helper and amplifier cells. Thus, CD4, CD45RA+, CD30+ T helper cells were given the designation of Th2 cells. These cells are resting cells with a life span in the peripheral blood of 5 -7 weeks. Th2 cells produce a pattern of cytokines that accelerates antibody production (especially IgE) and enhances eosinophil production (Mossman and Sad, 1996). Elevated numbers of Th2 cells are often found in disease states such as asthma, allergic responses, and helminth infections. Although it is known that Th2 cells play a critical role in defense against helminths, it is unclear whether elevated Th2 cells in allergic diseases are a cause or a consequence of the disease (Table 1).

The CD4, CD45RO+, CD30- amplifier population was given the designation Thl cells. These cells are long lived in comparison to Th2 cells. The extended life span ofThl cells may be the basis for immunological memory. Thl

Table 1. Association of Th2 Cells with Specific Disease States


Trichuris muris Nippostrongylus brasiliensis Heligimosomoidespolygyrus Brugia malayi Schistosoma mansoni

Th2 response in resistant mice cures

Th2 response associated with rejection Th2 response associated with expulsion Th2 response correlates with protection Th2 associated with granulomas


Borrelia burgdorferi

Th2 cells confer resistance


Immediate allergy

Th2 detected in late asthma and allergen-specific T-cell clones

Modified from Immunology Today, Mossman and Sad, 1996, vol. 17:138—45, with permission of Elsevier Science.

cells activate cytotoxic CD8 T cells and mediate inflammatory responses and delayed hypersensitivity reactions (Mossman and Sad, 1996) Thus, the Thl amplifier cells are elevated in tuberculosis, tuberculoid leprosy, and Listeria monocytogenes and Candida albicans infections (Table 2).

Presently there are no reagents separating the T cytotoxic and T suppressor subsets. In reality, the designation of T suppressor cells may be a misnomer as many T cell subsets can suppress reactivity by the secretion of various cytokines. Subsets of CD8 cells are identified by the expression of the CD28 marker. Cytotoxic T cells involved in tumor lysis are CD8+, CD28-. A second population of CD8 cells expressing the CD28 protein is weakly cytotoxic and secretes a potent antiviral protein.

There are several B cell subpopulations. Antibody-producing B cells have receptors for complement components C3d (CD21) and C3b (CD35) and recep-

Table 2. Association ofThl Cells with Specific Disease States


Leishmania major L. donovani L. brasiliensis

Thl cures and confers resistance Thl cures local leishmaniasis Thl localized disease


Candida albicans

Thl response correlates with resistance


Mycobacterium tuberculosis M. leprae

Thl correlates with resistance

Thl found at site of delayed hypersensitivity reactions

Modified from Immunology Today, Mossman and Sad, 1996, 17:138—45,with permission ofElsevier Science.

tors for antibodies (CD32). Interaction of ligands with the receptors stimulates the B cells to differentiate into plasma cells that produce protein antibodies. B cells constitute approximately 20% of the circulating lymphocyte population and 40% of the splenic lymphocytes.

A second population of B cells, called B-l cells, localize in the peritoneum and pleural cavities. They are distinguished from the normal B cell pool by several characteristics including the expression of both T and B cell markers. They appear early in ontogeny and are capable of self-renewal without bone marrow promotion. Generally, B-l cells produce IgM, IgA, and IgG antibodies with low affinity and broad specificity for bacterial antigens such as phospho-rylcholine, lipopolysaccharide endotoxins, and a 1-3 dextrans. These molecules are major components of the gram-negative bacterial cell wall.

Because of the localization in the gut and the production of antibacterial antibodies, B-l cells are important in mucosal immunity. In addition, they produce antibodies reactive with thyroglobulin and single-stranded DNA. Clearance of denatured self antigens inautoimmune diseases such as Sjogren's syndrome or systemic lupus erythematosus (SLE) may be the major function of antibodies produced by B-l cells.

There is a third population of lymphocytes. These cells are neither T nor B cells. Rather, they represent a unique lineage with specific effector functions. Because cells of this lineage lyse tumor cells without prior sensitization, they are called natural killer cells. NK cells are more difficult to enumerate by flow cytometry because they express markers found on both T and B cells. However, antibodies directed toward the CD16 and CD38 markers have been used to identify the NK cell population. The subset of NK cells that actually lyse cells from solid tumors express both the CD16 and the NKH1 marker (Storkus and Dawson, 1991).

Lymphocyte Development and Maturation. During ontogenesis in mammals, hematopoiesis occurs first in the yolk sac and the fetal liver. When the bone marrow develops, hematopoiesis shifts to the marrow. The maturation of stem cells in the marrow requires a stable microenvironment of reticular cells (adventitial and fibroblastic), adipocytes, endothelial cells, macrophages, and T cells (Mayani et al., 1992; Weiss and Geduldig, 1991). The maturation of these cells requires a complex interaction of cytokines and lineage-specific growth factors (Fletcher and Williams, 1992; Kincade, 1992). Rodents are the exception to the rule. Hematopoiesis also occurs in the red pulp of the spleen and the thymus. In the adult mammal, over 95% of hematopoiesis occurs in the bone marrow (Mayani et al., 1992). However, the distribution of the stem cells within the marrow differs according to species. All extramedullar space in the rat marrow is occupied with hematopoietic stem cells. In contrast, similar tissue in the dog, human, and rabbit is localized in the epiphysis of long bones, the skull, and the central skeleton.

T Cell Development. The thymus is the master organ of the immune system. It receives lymphocyte precursors from the bone marrow and stimulates growth, maturation, and differentiation of select CD4 and CD8 lymphocyte subsets. Immature lymphocyte precursors from the bone marrow enter the cortex of the thymus where they take on the appearance of large lymphoblasts. Approximately 5% of the resting cells do not express CD4 and CD8 markers (CD4-, CD8-). Another population of double-negative cells (80%) is rapidly dividing and rearranging TCR genes to develop a complete repertoire of antigenic recognition (Fig. 1). IL-7 producedby stromal cells initiates an irreversible transition process. Although the exact intrathymic effects are unclear, most hormones of the thymus also modulate the maturation of T cells. These hormones are thymulin, thymopoietin, thymic humoral factor (THF), and thymosins (Pawlowski and Staerz, 1994).

Bacteria Effector Into Cells

Figure 1. The maturational pathway of T cells in the thymus followed by most T cells expressing the aP T-cell receptor (TCR). Transition from the CD25+ double-negative cells to single-labeled cells requires the synthesis of P chain protein and activation of the protein tyrosine kinase p56Ick. Modified from Immunology Today, Anderson and Perlmutter, 1995, 16:99-105, with permission of Elsevier Science.

Figure 1. The maturational pathway of T cells in the thymus followed by most T cells expressing the aP T-cell receptor (TCR). Transition from the CD25+ double-negative cells to single-labeled cells requires the synthesis of P chain protein and activation of the protein tyrosine kinase p56Ick. Modified from Immunology Today, Anderson and Perlmutter, 1995, 16:99-105, with permission of Elsevier Science.

Normal transition of the double-negative cells requires the expression of a CD25 molecule, the production of a TCR B chain protein, and the expression and activation of p56 Ick that allows intracellular signaling. However, the p56 Ick signaling process is unique to thymocyte development. Neither p59 Ick nor p59iyn, which are closely related to p56 Ick in structure and function, increase activity during the T cell transition process. Moreover, generalized inhibition of the common FTK-mediated growth pathway does not inhibit p56 Ick activity. Most evidence supports the hypothesis that' p56 Ick serves to control the recombination of the variable portions of the MHC 11 Va and VB regions of the TCR.

Under the influence of thymic hormones produced by epithelial cells, the double-negative cells begin to mature over a period of 2 or 3 weeks. Initially, proliferating cells express low levels of CD8. Most cells transition into doublepositive (CD4+, CD8+) cells. Such cells constitute over 80% of the total cells in the adult thymus. As the double-positive cells move from the cortex to the thymic medulla, TCR a is also expressed on the cell surface. At this point, the CD3 marker is upregulated and expressed on the lymphocytes (Fig 1). About 50% of the cells are CD3+,TCR a/p and halfare CD3-TCR a/p .

To develop the complete repertoire of antigenic specificities, TCR a and B chains undergo random rearrangements. To ensure that cells leaving the thymus are capable of MHC-restricted expansion but unable to react with host tissue, a small proportion of double-staining cells have low binding affinity for peptide/ self MHC complexes expressed on stromal cells, fibroblasts, or thymic epithelial cells. These dual-staining cells are stimulated to further differentiate (positive selection). The purpose of positive selection is twofold. First, selection is required for additional T cell maturation. Second, interactions with MHC bias the T cell repertoire to self-restricted expansion. The remaining unselected population containing the autoreactive cells or T cells with high-affinity TCR for MHC has a short life span of 3-4days and undergoes negative selection by programmed cell death or apoptosis (Pawlowski and Staerz, 1994).

At 12 days of the maturation process, CD4+, CD8- TCR a/p represent 1215% of the total thymocytes. These cells ultimately evolve into helper cells with class II MHC-restricted activity. Because these cells have been processed by the thymus, they are commonly called Thelper cells. By day 19, CD4-,CD8+TCR a/p cells appear in the thymus. Although these cells comprise only 3% of the total thymocytes, they are released into the peripheral blood in high numbers to become class I MHC-restricted T CD8 cytotoxic cells.

There is a distinct, second lineage of a/p cells. In the thymus, a small population (5%) carries a CD4-, CD8- TCR a/p phenotype. Although these cells are often autoreactive, they can exit the thymus without undergoing positive or negative selection. Because these cells are processed differently than the conventional thymocytes, it has been speculated that these T cells have an alter native function in defense and are most efficient in recognizing self peptides presented by MHC proteins (Anderson and Perlmutter, 1995).

6 Cell Development. In chickens, a gut-associated lymphoid organ called the bursa of Fabricius is the sole source of B cells and antibody-producing plasma cells. Extirpation of the bursa causes a severe defect in the plasma cells resulting in reduced levels of immunoglobulins in the serum. However, bursec-tomized chickens have normal T cell immunity. Located in the hindgut (cloaca), the bursa is anatomically similar to the thymus. However, the bursa is packed with plasma cells rather than small T lymphocytes. Like the thymus, the bursa undergoes atrophy when the chicken undergoes puberty (4-6 weeks of age). Many immunologists have suggested that mammalian intestinal Peyer's patches (PPs) and other gut-associated lymphoid tissue are analogous in function to the bursa of Fabricius.

In mammals, the role of the gut-associated lymphoid tissue in B cell development is unclear and may be species dependent. In rabbits, there is no evidence that PPs, appendix, or sacculus rotundus play a role in B cell differentiation (Cooper and Lawton, 1972). Rabbit gut lymphoid tissue contains only the precursors of IgA-producing B cells that migrate to other areas of the gut following antigen stimulation (Craig and Cebra, 1971). In this species, there is a consensus that the bone marrow is the sole source of B cells (Cooper and Lawton, 1972). In other species, PPs are the primary source of B cell differentiation. In the sheep, physiological and anatomic data show that PPs function as a primary lymphoid organ (Reynolds et al., 1991) and provide the necessary microenvironment for B cell proliferation and antigen-driven mutation of immunoglobulin genes (Rey-naud etal., 1988) .

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