CHAPTER 82 LYMPHOCYTE ONTOGENY
CHAPTER 82 LYMPHOCYTE ONTOGENY
TUCKER W. LEBIEN
FRED E. BERTRAND
Lymphocyte Development from Pluripotential Stem Cells
Organs Involved in B-Cell Development
Immunoglobulin Gene Rearrangement and Expression
Expression of Recombination-Associated Genes
B-Lineage–Associated Surface Antigens
Regulation of B-Cell Precursor Growth
Organs Involved in T-Cell Development
T-Cell Receptor Gene Rearrangement and Expression
Expression of Recombination-Associated Genes
T-Lineage–Associated Surface Antigens
Regulation of T-Cell Precursor/Thymocyte Growth
Organs Involved in NK-Cell Development
NK Receptors and Recombination-Associated Genes
NK-Lineage–Associated Surface Antigens
Regulation of NK-Cell Growth
The functional mammalian immune system consists of three major lymphocyte populations with different antigen recognition systems: thymus-derived (T) cells; bursal, or marrow-derived, (B) cells; and natural killer (NK) cells. The three populations mediate complex and distinct immune effector functions. The development and manifestation of immune effector functions reflect differences in patterns of gene expression in T, B, and NK cells. Of primary importance in T and B cells are receptor complexes that mediate antigen recognition: the T-cell receptor (TCR) on T cells and the B-cell receptor (BCR) on B cells. NK cells do not express antigen-specific receptors; rather, specificity of NK cell recognition is provided by inhibitory signals transduced by receptors recognizing class I major histocompatibility complex molecules.1 The differences in effector functions and organ distribution can be traced to differences in early ontogeny. This chapter reviews lymphocyte ontogeny. We emphasize the genesis, gene expression, developmental options, and growth requirements of T, B, and NK cells. A more extensive discussion of B-cell, T-cell, and NK-cell function can be found in Chap. 83, Chap. 84, and Chap. 85, respectively.
Acronyms and abbreviations that appear in this chapter include: B, marrow derived; BCR, B-cell receptor; CD, cluster of differentiation; CS-1, connecting segment 1; DC, dendritic cell; DP, double positive; FL, flt-3 ligand; Ig, immunoglobulin; IL, interleukin; KIR, killer inhibitory receptors; NK, natural killer; PSC, pluripotential stem cells; RAG, recombination activating gene; yLC, surrogate light chain; SP, single positive; T, thymus derived; TCR, T-cell receptor; TdT, terminal deoxynucleotidyl transferase; TREC, TCR rearrangement excision circle; TRI, trilineage; VCAM-1, vascular adhesion molecule-1.
LYMPHOCYTE DEVELOPMENT FROM PLURIPOTENTIAL STEM CELLS
Lymphocytes are derived from pluripotential stem cells (PSC), also called hematopoietic stem cells. Pluripotential stem cells have the capacity to give rise to all cellular components of the blood and are discussed in greater detail in Chap. 14. Two terms used to describe the potential fate of PSC are self-renewal and differentiation. Self-renewal refers to the capacity of a cell to divide and give rise to two daughter cells that exhibit an indistinguishable pattern of gene expression. Differentiation refers to the genetically programmed sequence of events wherein cells undergo orderly changes in gene expression, culminating in a precursor-progeny relationship. The closer two cells are in a developmental pathway, the subtler are the differences in gene expression.
For many years the embryonic yolk sac (i.e., tissue outside the embryo proper) was considered to be the reservoir of self-renewing PSC that contribute to life-long lymphohematopoiesis.2 That concept has been seriously challenged by studies in murine embryos indicating that intraembryonic tissue encompassing paraaortic splanchnopleura and derived aorta-gonad-mesonephros harbor PSC prior to the fetal liver.3,4 Furthermore, lymphoid precursors were identified in the paraaortic splanchnopleura prior to the onset of circulation.3,5 Human hematopoiesis initiates in the yolk sac at week 3 of gestation and shifts to fetal liver circa week 5.6,7 Two studies employing human embryonic tissue addressed whether an intraembryonic source of definitive human PSC exists. The cell surface sialomucin CD34, a widely used marker for human PSC (see Chap. 14), was used to identify candidate stem cells. In one study, highly proliferating CD34+ stem cells were identified in 5-week human embryonic tissue devoid of yolk sac and liver anlage.8 In a second study, clusters of CD34+ hematopoietic-like cells were detected in the ventral aortic endothelium.9 CD34+ hematopoietic cells clustered in the ventral aortic endothelium were analyzed by the technique of in situ hybridization and were shown to express transcription factors and growth factor receptors important in early hematopoiesis.10 It is conceivable, however, that CD34+ PSC migrate from the yolk sac or ventral aortic endothelium to the fetal liver and differentiate into lymphoid progenitors with multilymphoid lineage potential.
Landmark studies in the mouse employing chromosomal markers11 and transfected gene markers12 were instrumental in defining the relationship between PSC and cells committed to the T, B, and NK lineages. Antibodies to specific cell-surface antigens and fluorescence-activated cell sorting were used to purify mouse PSC.13 Mouse PSC express cell-surface Sca-1 and low levels of Thy-1 but are negative for lineage-specific antigens. Cells with this phenotype constitute less than 0.05 percent of adult mouse marrow. Fewer than 100 of these cells injected into an irradiated mouse can reconstitute the entire lymphohematopoietic system, including B and T cells.13,14
The development of T, B, and NK cells from PSC is well accepted, but the existence of a common lymphoid progenitor that is restricted to developing into T, B, or NK cells, but not myeloid and erythroid cells, has been controversial. Data from the study of alterations in the hypoxanthine guanine phosphoribosyl-transferase gene in blood mononuclear cells from an atomic bomb survivor suggest that T, B, and NK cells are derived from a common progenitor.15 However, it is not clear whether the progenitor characterized in this study was truly lymphoid restricted or whether it also harbored the capacity to differentiate into nonlymphoid (i.e., myeloid and erythroid) blood cells. Two recent reports have provided fresh evidence supporting the existence of a common lymphoid progenitor in humans and mice. Fluorescence-activated cell sorting was used to isolate a rare population of CD10+/CD19– lymphoid progenitors from human adult and fetal marrow.16 These CD10+/CD19– cells were capable of developing into T, B, NK, or lymphoid dendritic cells, but not myeloid or erythroid cells.16 A second study used fluorescence-activated cell sorting to isolate a rare population of mouse marrow lymphoid cells expressing the interleukin-7 (LI-7) receptor and demonstrated that a single one of these cells could generate at least T and B cells, but not myeloid cells.17 However, single cell plating of individual fetal liver-derived mouse stem cells in vitro has suggested the existence of bipotential precursors with the capacity to mature into myeloid or B lineage cells.14 Thus, more work is necessary to fully characterize the common lymphoid progenitors and the signaling events (e.g., cytokines or stromal-cell–associated molecules) that regulate survival, growth, and apopt osis of these cells. Figure 82-1 shows the existence of common lymphoid progenitors in the marrow and the developmental potential of this progenitor. It is important to emphasize that the existence of this cell is based on a single study.16 The cell designated common lymphoid progenitors-TRI (for trilineage) is a candidate progenitor that migrates to the thymus (see below), but experimental evidence that this cell exists is lacking.
FIGURE 82-1 Developmental relationship among PSC, common lymphoid progenitors, and T, B, NK, and lymphoid DC lineages. The common lymphoid progenitors-TRI progenitor is proposed to be distinct from the common lymphoid progenitors by virtue of the inability of common lymphoid progenitors-TRI to develop into the B lineage. The two pathways shown for common lymphoid progenitors-TRI development in the thymus are based on a review of the literature discussed by Spits and colleagues.102 The NK/DC dual progenitor shown in pathway 2 is based on studies by Márquez et al.106 The dashed line underneath the PSC indicates the capacity to undergo self-renewal. The arrows with parallel slashes indicate that several stages of development between a progenitor (e.g., a pro-B cell) and a mature lymphocyte (e.g., a B cell) have been deleted for simplicity. Completed pathways for B, T, and NK development are shown in Fig. 82-2, Fig. 82-3, and Fig. 82-4.
FIGURE 82-2 B-cell development in human marrow. The common lymphoid progenitors correspond to the common lymphoid progenitors in Fig. 82-1 with multilymphoid lineage developmental potential. The TdT, RAG, VpreB, Iga, and µ designations within the cells indicate nuclear (TdT and RAG) and cytoplasmic (VpreB, Iga, and µ) expression of these proteins. The shaded areas indicate active Ig gene rearrangement at that stage of development. Marrow stromal cells is a generic phrase encompassing several different nonlymphohematopoietic cell types in the marrow microenvironment.
FIGURE 82-3 T-cell development in human thymus. Two potential progenitors of the pro-T cell are shown: T/NK and common lymphoid progenitors-TRI. These two progenitors reflect the two possible pathways shown in Fig. 82-1 wherein either a T/NK or a common lymphoid progenitors-TRI could be the immediate progenitor of the pro-T cell. The TdT and RAG designations indicate nuclear expression of these proteins. The shaded areas indicate active TCR rearrangement at that stage of development. Thymic stromal cells is a generic phrase encompassing several different cell types in the thymic microenvironment.
FIGURE 82-4 NK-cell development in human thymus or marrow. Three possible progenitors of the pre-NK cell are shown: common lymphoid progenitors, T/NK, and NK/DC. The common lymphoid progenitors correspond to the common lymphoid progenitors in Fig. 82-1 with multilymphoid lineage developmental potential. The common lymphoid progenitors is a marrow progenitor of the pre-NK cell based on studies by Galy et al.16 The T/NK is an intrathymic progenitor of the pre-NK cell based on studies summarized by Spits et al.102 The NK/DC is an intrathymic progenitor of the pre-NK cell based on studies by Márquez et al.106 Two populations of mature NK cells are shown that circulate in the blood (see the text).
The defining characteristic of a mature B cell is the expression of cell surface immunoglobulin (Ig). The cell-surface Ig consists of mu (µ), delta (d), gamma (g), alpha (a), or epsilon (e) heavy chains disulfide-linked to kappa (k) or lambda (l) light chains (see Chap. 83). The cell surface Ig and associated signaling molecules (see below) are often referred to as the BCR. Precursor (pre-) B cells are generally defined by the presence of cytoplasmic µ heavy chains in the absence of cell surface Ig. Progenitor (pro-) B cells are generally defined by the absence of cytoplasmic and surface µ heavy chains. This minimalist definition of pro-B, pre-B, and B cells18,19 forms the basis of the present detailed model of human B-cell development. B-cell development can be divided into two stages: an antigen-independent stage that occurs primarily in fetal liver and fetal and adult marrow, and an antigen-dependent stage that occurs primarily in secondary lymphoid tissue, such as spleen and lymph node.
ORGANS INVOLVED IN B-CELL DEVELOPMENT
Human fetal liver and omentum are the main sites of B-cell development from 8 to 14 weeks’ gestation. Fetal omentum is a thin, vascularized, membranous fold of peritoneum and has been considered part of the lymphoid system because it contains aggregates of lymphoid cells called milk spots. Pre-B cells can be detected in fetal liver at 7 to 8 weeks’ gestation18 and in fetal omentum at 10 weeks’ gestation.20 Second-trimester B-cell development is multifocal, since pro-B, pre-B, and immature B cells can be detected in the marrow and liver and, to a lesser extent, the lung and kidney.21 From the end of the second trimester throughout adult life, marrow is the exclusive site of B-cell development. The frequency of early B-lineage cells as a percentage of the total nucleated lymphohematopoietic cell pool is much higher in fetal than in adult marrow.21,22
IMMUNOGLOBULIN GENE REARRANGEMENT AND EXPRESSION
The hallmark of marrow B-cell development is the ordered rearrangement of gene segments that encode the variable portion of the antibody molecule (see Chap. 83 for additional detail). This process typically begins with germ-line transcription of the µ heavy-chain locus, followed by the joining of one of 30 DH segments to one of six JH segments.23 Once this DJH join is completed, one of approximately 130 upstream VH gene segments24 can join to form a completed VDJH rearrangement.23 If the VDJH rearrangement is “in frame,” meaning that it is capable of encoding a functional µ heavy-chain protein, VDJ rearrangement ceases. This process is termed allelic exclusion. Experiments in mice demonstrated that allelic exclusion depends on the presence of µ heavy chain,25 but the mechanism or mechanisms of allelic exclusion is unknown. If the initial VDJH rearrangement is nonfunctional (i.e., the rearrangement does not encode a µ heavy-chain protein), then rearrangement can proceed to the other heavy-chain allele. If a nonfunctional VDJH rearrangement occurs on both alleles, the cell undergoes apoptosis and is phagocytized by marrow macrophages.
Following completion of a functional µ heavy-chain rearrangement, recombination at the light-chain locus proceeds in a similar fashion.23 This usually begins with germ-line transcription of the k locus followed by the rearrangement of a Vk to Jk. If an initial VJk rearrangement is functional, the light-chain locus also undergoes allelic exclusion. If not, rearrangement proceeds to the other k allele. It is generally thought that, should recombination at both k alleles fail to result in a functional rearrangement, the l light-chain locus will rearrange.23 An alternative model proposes that light-chain rearrangement begins at the k and l loci simultaneously.26 Due to a difference in recombination rate, the k locus usually finishes recombining first, potentially explaining the high ratio of k- to l-expressing B cells in the mouse.26
Heavy- and light-chain Ig gene rearrangement encompasses multiple stages of human B-cell development, shown in Fig. 82-2. DJH rearrangements initially occur in a CD10+/CD34+/CD19– progenitor designated the early B cell.27,28 CD19+/CD34+ pro-B cells harbor VDJH rearrangements,27 and a functional VDJH rearrangement results in differentiation to the pre-BI cell stage. The pre-BI compartment is enriched for cells in cycle that express the pre-BCR (see below). The subsequent pre-BII compartment undergoes V to Jk light-chain rearrangement, and pre-BII cells with functional light-chain rearrangements differentiate into immature B cells expressing µ/k or µ/l BCR. Ig gene rearrangement generally proceeds through an ordered progression, beginning with the µ heavy-chain gene and proceeding through k and (if necessary) l light-chain genes.29 Alternative pathways have been described in which light-chain rearrangement precedes heavy-chain rearrangement30 and l light-chain rearrangement precedes k light-chain rearrangment.31
At the pro-B to pre-B transition, µ heavy-chain encoded by a functional VDJ rearrangement associates with a complex called the surrogate light chain (yLC).32 yLC is a heterodimeric protein complex consisting of the l5 and VpreB gene products.33,34 yLC genes exhibit significant homology with conventional l light chains but do not undergo gene rearrangement. The µ-yLC receptor associates with the Iga and Igb signaling molecules (see below) to form the pre-BCR.32 The VpreB protein is expressed in the cytoplasm of CD19– early B cells prior to the expression of µ heavy chain.35 However, surface expression of the µ-yLC pre-BCR is restricted to a subset of large pre-BI and small pre-BII cells.35,36
The importance of the yLC in B-cell development was first demonstrated in mice with a targeted disruption in the l5 locus.37 l5-deficient mice exhibit a block in B-cell development at the pro-B to pre-B transition, presumably because the cells fail to receive a positive selection signal in the absence of a functional pre-BCR.37 Patients with mutations in the l5 gene exhibit a profound block in pro-B to pre-B cell differentiation,38 confirming the importance of the µ-yLC in human B-cell development as well.
Pre-B cells undergo a type of positive selection by virtue of pre-BCR expression. That is, pre-BCR expression is a requisite for pre-B cell survival and differentiation. A ligand for the pre-BCR has never been identified, and the molecular basis for this positive selection is unknown. However, recent studies in the mouse provide a clue. Some nascent µ heavy chains can physically pair with yLC, whereas others cannot. Pre-B cells expressing µ heavy chains that can pair with yLC to form a pre-BCR will survive and differentiate into immature B cells.39,40
EXPRESSION OF RECOMBINATION-ASSOCIATED GENES
Two genes, designated recombination activating gene-1 (RAG-1) and recombination activating gene-2 (RAG-2), are necessary and essential for Ig and TCR gene rearrangements.41 Mice deficient in either RAG-1 or RAG-2 have severe disruptions in early B-cell development.42,43 Furthermore, mutations in the RAG genes have been identified in some patients with severe combined immunodeficiency.44 In vitro studies have shown that the RAG gene products bind to recombination signal sequences and introduce double-stranded DNA breaks during initiation of the recombination cleavage reaction.45 In humans, RAG-1 and RAG-2 are expressed from the early-B-cell stage through the pre-B stage of development (Fig. 82-2). Once a cell has successfully completed heavy- and light-chain rearrangement, RAG gene expression generally ceases. In B cells with autoreactive BCR specificity, RAG expression can be reactivated by a process called receptor editing.46,47 Little is known about how expression of RAG genes is regulated. Some investigators have reported that levels of RAG transcription vary as a function of B-cell development.29 In addition, the RAG-2 protein is regulated in a cell-cycle-dependent fashion through phosphorylation.48 The promoters for human RAG-1 and RAG-2 have been identified.49 Although the promoter sequences will drive transcription of each gene, they are not sufficient to confer lineage and stage specificity to RAG transcription.
Terminal deoxynucleotidyl transferase (TdT) is not required for the recombination reaction but is necessary for the generation of antigen-receptor diversity. TdT inserts non–template-encoded nucleotides (so-called N-region insertions) at the DH to JH and VH to DJH junctions of heavy chains.50 The N-region insertions serve to increase the diversity of the antigen-receptor repertoire to encode more than 1 × 108 different specificities. A limited number of k light-chain rearrangements contain TdT N-region insertions.51 Mice bearing a targeted deletion of the TdT gene lack characteristic N-region insertions at the DH to JH and VH to DJH heavy-chain boundaries.52,53 TdT protein is initially expressed in common lymphoid progenitors.15,54 As shown in Fig. 82-2, the CD19+/CD34+ pro-B–cell population is TdT+, but expression is turned off in the pre-B cell.29,55,56 Reduction in TdT expression at the pro-B to pre-B transition explains why light-chain gene rearrangements have so few N-region insertions.
B-LINEAGE–ASSOCIATED SURFACE ANTIGENS
Although Ig gene rearrangement accords the most precise molecular definition of B-cell development, monoclonal antibodies recognizing cell surface molecules have been essential for characterizing and isolating specific stages by flow cytometry and cell sorting.57 Figure 82-2 shows the expression of several cell surface molecules that have been useful in characterizing different stages of human B-cell development. For a broader consideration of the CD antigens, see Chap. 13. Analysis of lymphoid cells with a CD10+/CD34+/CD19– cell-surface phenotype reveals expression of cytoplasmic VpreB35 and DJH rearrangements27,28 in some of these cells, which may comprise an early-B-cell compartment. A compartment designated the pro-B cell is characterized by expression of cell surface CD10, CD34, and the B-lineage–restricted molecule CD19. A small percentage (»10%) of pro-B cells express the VpreB component of the pre-BCR on the cell surface,35 but it is not known whether this CD19+/CD34+/VpreB+ population expresses surface µ heavy chain or whether VpreB is associated with other molecules.58 The next major compartment is the pre-B cell, characterized by the expression of CD19, the loss of CD34 and TdT, and the acquisition of µ heavy chain in more than 90 percent of the cells.29,55,56 The pre-B–cell population can be subdivided into large cycling cells and small noncycling cells based on cell cycle analysis.29 A subset of pre-B cells expresses the µ-yLC pre-BCR29,35 in association with a heterodimer designated Iga/CD79a and Igb/CD79b. The Iga/Igb heterodimer is an essential molecular complex that initiates signal transduction following cross-linking of the pre-BCR and BCR.59,60 It is interesting to note that Iga and Igb are expressed in CD10+/CD19– early B cells prior to the appearance of functional VDJH rearrangements.61 The function of Iga and Igb in early B cells is unknown. Mice with a targeted disruption of the Igb gene are blocked at the level of DJH rearrangement,62 suggesting a critical function for the Igb protein separate from its functional role in the cell surface µ-yLC pre-BCR. Once pre-B cells successfully rearrange k or l light chain genes, they differentiate into immature B cells expressing the BCR. Immature B cells also express high levels of B-cell–restricted cell-surface molecules, including CD20, CD21, CD22, and CD40.
REGULATION OF B-CELL PRECURSOR GROWTH
B-cell development in marrow is regulated by a complex interplay of signals transduced by stromal cells, the extracellular matrix (e.g., fibronectin and type IV collagen), and possibly other lymphohematopoietic cells. The continuous production of B cells throughout life21 is dependent upon an intact marrow microenvironment.
The interaction or adhesion of human B-cell precursors to the marrow stromal cell microenvironment is mediated through VLA-4 and VLA-5 integrins.63,64 VLA-4 has two ligands: vascular cell adhesion molecule-1 (VCAM-1) and the CS-1 domain of fibronectin.65 The ligand for VLA-5 is the central cell-binding domain of fibronectin that contains the amino acid sequence arginine-glycine-aspartic acid, or RGD.65 Marrow stromal cells constitutively express VCAM-1.63,64 Functional expression of VCAM-1 can be positively regulated by IL-1b and IL-4 and negatively regulated by TGF-b.64 VLA-4/VCAM-1 interaction is critical for the adhesion of B-cell precursors to marrow stromal cells, but the function of VLA-4 in B-cell precursors beyond simple adhesion is not well understood.
Human B-cell precursors can be positively or negatively influenced by several different cytokines.66 IL-7 facilitates the marrow stromal cell–dependent growth of B-cell precursors.67 The CD19+/CD34+ pro-B cell is the IL-7–responsive population that grows on marrow stromal cells,68 and IL-7 triggers decreased expression of RAG-1, RAG-2, and TdT.68,69 A combination of IL-7, IL-3, and the flt-3 ligand (FL) is the strongest known stimulus for the growth of human pro-B cells.70 A striking species difference characterizes the requirement for IL-7 signaling in human and murine B-cell development. Murine B-cell development has an absolute requirement for IL-7–IL-7-receptor interaction and subsequent downstream signaling involving the gc subunit of the IL-7 receptor and the Jak-3 tyrosine kinase.71 In contrast, IL-7 is necessary but not sufficient for human B-cell development. X-linked severe combined immunodeficiency patients have mutations in the gc cytokine–receptor subunit and exhibit profound thymic hypoplasia, an absence of NK cells, but normal or elevated numbers of B cells.72 Immunodeficiency patients with mutations in Jak-373,74 or the IL-7 receptor75 also have normal numbers of blood B cells. Furthermore, an in vitro model of human B-cell development is IL-7 independent.76 These collective results indicate that IL-7 is not e ssential for at least the numerically normal development of human B cells. The identity of the human marrow stromal cell-derived molecule or molecules that provide the essential proliferative stimulus for human pro-B cells is unknown.
As discussed above, B-cell development can be detected in week 15 human fetal marrow, and the marrow is the primary source of B-cell development throughout life.21 A fundamental distinction between mammalian B- and T-cell development is that the latter generally consists of an essential progenitor migration step from fetal liver or fetal or postnatal marrow to the thymus. Thus, PSC in fetal liver and marrow or postnatal marrow may differentiate into a progenitor that migrates to the thymus (see Fig. 82-1). The precise phenotype of this migratory progenitor is unknown. Relatively little is known regarding the mechanisms that attract marrow lymphoid precursors to the thymus, but chemotactic factors (e.g., chemokines) produced by thymic stromal cells probably play a role.77
ORGANS INVOLVED IN T-CELL DEVELOPMENT
FETAL LIVER AND MARROW
Studies of human T-cell lymphoblastic leukemia revealed the CD7 cell surface molecule as a candidate marker of early T-cell development.78 In support of this concept, CD7+ lymphoid cells expressing cytoplasmic CD3e are present in fetal liver at 7 to 8 weeks’ gestation.79,80 Furthermore, CD7+/cytoplasmic CD3e+ progenitors are present in fetal liver and fetal thorax just prior to colonization of the epithelial thymic rudiment.79 Rare CD34+/CD7+ lymphoid cells are also present in fetal marrow.81 One difficulty in interpreting the collective data in these studies is that CD7 is also expressed on NK progenitors and some myeloid cells and is therefore not a T-lineage–specific marker. The developmental relationship between cells that express CD7+ and cytoplasmic CD3e+ cells versus cells that express CD10 and the IL-7 receptor is unknown (see Fig. 82-1). However, the common lymphoid progenitors contain approximately 10 percent CD7+ cells.16 Thus, the CD7+/cytoplasmic CD3e+ cells may be comparable to the common lymphoid progenitors-TRI (see Fig. 82-1). To complicate matters, an alternative pathway of T-cell development that occurs in marrow has been described in the mouse.82 Whether a T-cell developmental pathway exists in human marrow is unknown. However, extrathymic T-cell maturation may occur in human fetal liver83 and fetal intestine.84
The human thymic microenvironment begins to develop at approximately 4 weeks’ gestation84 and then undergoes at least three developmental phases.86 The first phase occurs between 4 and 8 weeks’ gestation and is characterized by endoderm- and ectoderm-derived thymic epithelial cell proliferation. The second phase occurs between 9 and 15 weeks’ gestation and is characterized by the appearance of subcapsular, cortical, and medullary regions. Colonization by fetal liver–derived progenitors begins at about week 9. The third phase occurs from 16 weeks’ gestation until 1 to 2 years of age and is characterized by maximal intrathymic T-cell maturation. Historical views have assumed that the thymus only functions in young humans and mice because of its well-known involution during early life.87 This viewpoint has been seriously challenged by the use of a sophisticated polymerase chain reaction assay that detects extrachromosomal DNA circles called TCR rearrangement excision circles, or TRECs.88 TRECs are a product of TCR gene rearrangement and represent a molecular marker of thymic function. Using the TREC assay, thymic function was observed to decline by 5-fold at age 35 and 50-fold by age 65, but individuals over 70 years still had TRECs in their blood T cells.88 The T-cell pool appears to be replenished via a functional thymus (albeit in an age-dependent manner) throughout life.
Thymic T-cell maturation is an extraordinarily complex series of events that results in the development of a functional T-cell repertoire.89 In addition to the thymocytes, which are at multiple stages of development based on immunologic phenotype (see below), numerous nonlymphoid cells make up the thymic microenvironment.90 The subcapsular, cortical, and medullary regions of the thymus contain heterogeneous populations of epithelial cells, macrophages, and dendritic/interdigitating cells (see Chap. 5). Current models suggest that positive selection (the process by which T cells recognizing foreign peptides are selected) and negative selection (the process by which T cells recognizing self-peptides are eliminated) occur when developing thymocytes interact with thymic epithelial cells and dendritic/interdigitating cells, respectively. The heterogeneity of the epithelial cell compartment in the thymus is particularly striking,91,92 and the significance of this heterogeneity in the context of positive selection and other events in thymic T-cell maturation remains to be clarified.
T-CELL RECEPTOR GENE REARRANGEMENT AND EXPRESSION
Like the Ig molecule, the TCR is encoded by distinct gene segments (V, D, J, and C) that rearrange during T-cell development.93 TCR genes encode polypeptides designated a, b, g, and d (see Chap. 84).93 The a and b proteins pair with one another to form the TCRab. The g and d proteins pair with one another to form the TCRgd. The TCRab and TCRgd are noncovalently associated with a family of proteins called the CD3 complex. CD3 performs a crucial role in signal transduction following TCR cross-linking, analogous to the role of Iga/Igb in mediating signal transduction following BCR cross-linking. A more detailed description of TCR genes and proteins is presented in Chap. 84.
Rearrangement of TCRg and d genes occurs prior to rearrangement of TCRb and a genes during human T-cell development.94,95 The thymocyte compartment containing TCRg and d rearrangements with most TCRb genes in germ-line configuration is the pro-T cell (Fig. 82-3). TCRb rearrangements are initiated in the next stage of T-cell development: the early single-positive (SP) CD4+/CD8– thymocytes. If the TCRb rearrangement is functional, the encoded TCRb protein assembles into a complex called the pre-TCR. Cells expressing the pre-TCR are designated early double-positive (DP) thymocytes because they express both CD4 and CD8. If the TCRb rearrangement is nonfunctional, the early SP thymocytes undergo apoptosis. The elimination of developing T cells by this mechanism mirrors the elimination of pro-B cells that fail to make a functional VDJH rearrangement. The pre-TCR consists of TCRb protein, the CD3 signal transduction protein complex, and an invariant chain designated pTa. The pTa gene was originally cloned in the mouse96 and subsequently in humans.97,98 It is interesting to note that the pre-TCR functions as a sensor and facilitates the survival and expansion of pre-TCR+ thymocytes in a process called b selection.99 Rearrangement of TCRa genes subsequently occurs in intermediate DP thymocytes, eventually culminating in expression of low levels of surface TCRab in late DP thymocytes (see Fig. 82-3).
EXPRESSION OF RECOMBINATION-ASSOCIATED GENES
The role of RAG-1, RAG-2, and TdT in the rearrangement and diversification of the TCR repertoire is very similar to their role in rearrangement and diversification of the BCR repertoire. RAG-1- and RAG-2-deficient mice exhibit profound disruptions in normal thymocyte development due to a failure to initiate TCR rearrangement.42,43 Likewise, TdT-deficient mice lack N-region insertions and exhibit a restricted diversification of the TCR repertoire.52,53 RAG gene expression oscillates during murine thymocyte development, corresponding to two waves of rearrangement involving TCRb, g, d, and TCRa.100 Oscillation of RAG gene expression has not been reported in human thymocytes. Northern blot analysis of human thymocyte subpopulations indicated that RAG-1/RAG-2 were expressed in early SP thymocytes and all DP (CD4+/CD8+) thymocytes, but not CD4+ and CD8+ SP thymocytes.101
T-LINEAGE–ASSOCIATED SURFACE ANTIGENS
Studies during the last several years have provided direct evidence that the earliest definable lymphoid progenitors in the thymus are multipotential.102 Figure 82-1 shows the phenotype and developmental capacity of a thymic progenitor, the common lymphoid progenitors-TRI, that may be the direct migrant from fetal liver or fetal and postnatal marrow. The common lymphoid progenitors-TRI is CD34+/CD7+/IL-7 receptor-positive but does not express cell surface CD1a, CD2, CD3, CD4, CD5, or CD8.103 Several studies demonstrated that CD34+/CD7+/CD1a– thymic progenitors can develop into T, NK, and lymphoid dendritic cells.104,105 and 106 These studies provide direct evidence that marrow progenitors that migrate to the thymus are not developmentally restricted to become only T cells. It is interesting to note that preliminary studies indicate that the multipotential thymic progenitors cannot develop into B-lineage cells,102 suggesting that the marrow progenitor designated common lymphoid progenitors-TRI may be the cell that migrates to the thymus (see Fig. 82-1).
As shown in Fig. 82-3, acquisition of cell surface CD1 defines an important checkpoint in T-cell development, and the CD34+/CD1a+ pro-T cell population preferentially develops into the T lineage.102 The next developmental stage is the early SP thymocyte, which is CD4+/CD8–. Early SP thymocytes are fully committed to the T lineage and can give rise to TCRab- or TCRgd-expressing T cells.102 Early SP thymocytes differentiate into early DP thymocytes. Early DP thymocytes are easily distinguished from their early SP precursors by the expression of CD8 and the pre-TCR.101 Figure 83-3 shows three DP stages (early, intermediate, and late) that likely reflect a linear developmental pathway. Early DP thymocytes expressing the pre-TCR undergo substantial expansion in cell numbers. The pre-TCR is lost at the intermediate DP thymocyte stage, coincident with the onset of TCRa rearrangement and subsequent expression of low levels of TCRab (the late DP thymocyte). Dstinct subcompartments in the DP thymocyte pool have been identified.101 Late DP thymocytes differentiate into CD4+/CD8– SP and CD4–/CD8+ SP thymocytes expressing high levels of cell surface TCRab. These two populations correspond to helper/inducer and cytotoxic T cells that make up the blood and secondary lymphoid organ T-cell pool. They are discussed in more detail in Chap. 84.
REGULATION OF T-CELL PRECURSOR/THYMOCYTE GROWTH
Growth, differentiation, and apoptosis of thymocytes are regulated by direct physical interaction with thymic stromal cells (i.e., epithelial cells, fibroblasts, and dendritic/interdigitating cells) and cytokines produced by thymic stromal cells. Thymic epithelial cells synthesize and secrete a complex array of cytokines. These include IL-1, IL-3, IL-6, IL-7, stem cell factor, leukemia inhibitory factor, TGF-b, and several colony-stimulating factors.108,109,110,111 and 112 Cytokine stimulation probably occurs in the localized microenvironment of a thymic epithelial cell–thymocyte adhesive interaction. This may be accomplished through adhesive interactions mediated by thymocyte CD2 and CD11a interacting with thymic epithelial cell CD58 and CD54.113,114 Thymocytes also express VLA-4,81 and it is probable that thymic stromal cell VCAM-1 or fibronectin serves as the counterreceptor to facilitate adhesion.
What makes thymocytes grow? In contrast to human B-cell precursors, IL-7–IL-7-receptor interaction and downstream signaling events appear to be essential for normal human thymocyte development. Immunodeficiency patients with mutations in the gc subunit of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors72; patients with mutations in Jak-373,74; and patients with mutations in the IL-7 receptor75 all exhibit profound blocks in thymocyte development. The importance of IL-7 in these experiments of nature has been reproduced using a chimeric human-mouse fetal thymic organ culture.115 Exactly how IL-7 exerts its essential effect on human thymocytes is unknown, but IL-7 does activate phosphatidylinositol-3 kinase,116 a lipid kinase essential to many mitogenic pathways. Studies in the mouse may provide additional clues. IL-7 is a nonredundant cytokine that is essential for T-cell development.117 Mice deficient in expression of the IL-7 receptor a chain exhibit a severe block in thymocyte development, but this deficiency can be overcome by enforced expression of the antiapoptotic protein bcl-2.118,119 Furthermore, mice deficient in IL-7 show a profound loss of bcl-2 in immature thymocytes (i.e., CD3–/CD4–/CD8–), but short-term culture of these cells with IL-7 increases expression of bcl-2 and promotes cell survival.120 These collective results indicate that IL-7 functions to enhance bcl-2 levels in early thymocytes, thereby transmitting a survival signal essential for continuing thymocyte development. Whether human thymocyte survival is mediated by a similar IL-7-dependent mechanism is unknown.
NK cells are large granular lymphocytes that comprise approximately 5 percent of blood and splenic lymphocytes. NK cells play an important role in the innate immune response to infection and some tumors (see Chap. 85). Substantial progress in identifying and characterizing receptors on NK cells that transduce inhibitory signals has been made in the last several years.1 Due to the lack of NK-specific cell surface markers (see below), a rigorous analysis of NK-cell development has been difficult. However, with the increasing ability to isolate PSC and common lymphoid progenitors by fluorescence-activated cell sorting, coupled with the development of in vitro models for analyzing differentiation into lymphoid lineages, knowledge of NK-cell development has increased.
ORGANS INVOLVED IN NK-CELL DEVELOPMENT
NK-cell development can originate from CD34+ stem cells present in fetal liver, thymus, and marrow. However, it is unclear whether these three tissues generate discrete subsets of NK cells throughout life or whether they reflect a shift in the site of NK-cell development as a function of ontogeny. NK cells can be detected at 6 weeks’ gestation in fetal liver,121 prior to the development of the thymus as a functional organ that supports thymocyte maturation.85,86 Consistent with this ontogenic observation, fetal liver CD34+ stem cells can differentiate into NK cells in vitro.122 A close ontogenic relationship exists between NK cells and thymocytes.102 The development of NK cells from CD34+ thymic progenitors and the supportive capacity of the thymic microenvironment for NK-cell development provide strong evidence for a thymic origin of at least some NK cells.102,123,124 and 125 Thus, a T/NK progenitor is shown as pathway 1 in Fig. 82-1. A second pathway of thymic NK-cell development (see Fig. 82-1) is based on the isolation of a bipotential NK/DC progenitor characterized by high expression of the CD44 cell surface proteoglycan.126 CD34+ stem cell populations isolated from marrow and cord blood can develop into functional cytolytic NK cells using a variety of in vitro culture systems containing cytokines and stromal cells.127,128,129,130,131,132 and 133
NK RECEPTORS AND RECOMBINATION-ASSOCIATED GENES
Unlike T and B cells, NK-cell development does not require a process of receptor gene rearrangement mediated by RAG-1 and RAG-2. Thus, NK-cell development is essentially normal in mice deficient in RAG-1 or RAG-2.42,43 Human NK-cell recognition of target cells is mediated by two families of so-called inhibitory receptors that recognize polymorphic class I major histocompatibility complex molecules.1 These receptor families are known as CD94/NKG2 and killer inhibitory receptors (KIR).1 Following binding to class I, these inhibitory receptors transduce signals that culminate in the inhibition of NK-cell–mediated cytotoxicity and cytokine production. A more detailed discussion of these receptors can be found in Chap. 85.
NK-LINEAGE–ASSOCIATED SURFACE ANTIGENS
Analysis of NK-cell development has been compromised by the absence of NK-lineage–specific cell surface markers. Molecules expressed on the cell surface of NK cells, such as CD16, CD56, CD94, and the inhibitory receptors, are also variably expressed on T-lineage cells.1,102 Likewise, cell surface molecules commonly found on T cells, such as CD5, CD7, and CD28, are variably expressed on NK cells. The best way to distinguish T-lineage cells from NK cells is to assay for expression of the TCR, a complex found exclusively on T cells. Figure 82-4 shows a tentative scheme of NK-cell development.102,122,124,127,128,129,130,131,132,133 and 134 The developmental interface between any multilineage progenitor and the pre-NK cell is very poorly characterized. One of the earliest changes is the appearance of a molecule designated CD122, the b subunit of the IL-2/IL-15 receptors, on the pre-NK cell.133 Acquisition of CD56 defines the next stage of development, designated the immature NK cell. The mature NK-cell population (i.e., the population in blood) is composed of two subpopulations. The predominant subpopulation (»95% of mature NK cells) expresses CD16, low amounts of CD56, and both inhibitory receptor families (CD94 and KIR). The minority subpopulation (»5% of mature NK cells) expresses high levels of CD56 and CD94 but is KIR– and CD16–. There is no evidence that the CD16–/KIR– population is a precursor of the CD16+/KIR+ population,102 and it is also unclear whether the imm ature NK population can differentiate into the CD16–/KIR– population.
REGULATION OF NK-CELL GROWTH
NK cells, like B and T cells, require the complex milieu of the marrow or thymic microenvironment for growth and differentiation. A large number of cytokines have been shown to enhance the development of NK cells from CD34+ stem cells in vitro. These include IL-1a, IL-2, IL-6, IL-7, IL-15, FL, stem cell factor, and granulocyte-macrophage colony stimulating factor.122,123,126,127,128,129,130,131,132,133 and 134 Part of the difficulty in determining which cytokines are important reflects our current inability to distinguish common lymphoid progenitors from the earliest stages of NK development. Figure 82-4 portrays a pre-NK cell, but it is unclear whether a pre-NK cell is committed to the NK lineage. Moreover, the absence of NK-specific antigens has prohibited the use of fluorescence-activated cell sorting to purify NK progenitors. Thus, it is difficult to distinguish the effect of a given cytokine on PSC, common lymphoid progenitors, or an NK progenitor in the context of NK-cell development.
NK-cell development from CD34+ marrow stem cells has been a useful model for evaluating the contribution of marrow stromal cell products to NK-cell development. IL-2 can enhance NK-cell development,127,128,129 and 130 but IL-2 is only synthesized by antigen-specific T cells and is not produced by marrow stromal cells.132 IL-15 is a more plausible candidate. IL-15 binds to a receptor complex consisting of a unique a chain, a b chain (CD122) that is used by IL-15 and IL-2, and the common g chain (gc) used by IL-2, IL-4, IL-7, IL-9, and IL-15. Human marrow stromal cells produce IL-15, and IL-15 promotes the development of CD56+ NK cells from CD34+ marrow stem cells.132 FL also promotes the development of CD56+ NK cells from CD34+ marrow stem cells.131,133 A recent study suggests a potential hierarchical effect of FL and IL-15 on NK-cell development.133 FL was found to induce the expression of CD122 and IL-15 receptor a transcripts on CD34+ marrow stem cells. The FL-stimulated CD34+ stem cells could then respond to IL-15 and differentiate into CD56+/KIR+ NK cells with cytolytic activity.133 In another study, human thymic progenitors cultured in IL-15 gave rise to NK cells.132 These in vitro studies are consistent with experiments of nature. Severe combined immunodeficiency patients with mutations in the gc subunit or Jak-3 tyrosine kinase do not develop NK cells.72 As mentioned above, these mutations negate the function of IL-2, IL-4, IL-7, IL-9, and IL-15. IL-7 is not likely to be essential for NK-cell development, since patients with mutations in the IL-7 receptor have normal NK-cell development.75 IL-2 is not likely essential, as discussed above, and there is no known role for IL-4 or IL-9 in NK-cell development. However, IL-15 and IL-15 re ceptor a chain–deficient mice lack NK cells.135 These collective data indicate that IL-15 plays a pivotal role in NK-cell development. Definitive evidence for this in humans, however, may require the identification of an immune-deficient patient with a mutation in the IL-15 gene.
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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn