CELLULAR IMMUNITY OF THE HUMAN FETUS AND NEONATE

HIV AND WOMEN AND PREGNANCY

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CELLULAR IMMUNITY OF THE HUMAN FETUS AND NEONATE David B. Lewis, MD

T lymphocytes (T cells) have surface antigen-specific T cell receptors (TCRs) composed of either an alpha (a)and a beta (P) chain (Fig. l),or a gamma (y) and a delta (6) chain.31,39Both a/P- and yl6-TCR.s are always associated with CD3, a complex of four proteins that signal to the interior of the T cell after engagement of the TCR by antigen (Fig. l).Io3 The amino-terminal portion of each of the TCR chains is involved in antigen recognition and has a variable amino acid sequence. This variability is generated by rearrangement of TCR genes during T-cell development. The carboxy-terminal region is monomorphic or constant (C). The a/P-TCR recognizes protein antigens in the form of relatively short peptide fragments noncovalently bound to the groove of major histocompatibility complex (MHC) class I or class I1 molecules on the T cells bearing TCRs comantigen-presenting cell (APC) (Fig. 1)?o,127 posed of a and P subunits (a/P-T cells, for short) predominate in the thymus, circulation, lymph nodes and spleen. T cells bearing TCRs made of y and 6 chains (y/6-T cells) have a tissue distribution and antigen specificity that are distinct from those of a/P-T cells and are discussed separately. Class I MHC present peptides mainly to CD8 a/P-T cells. Virtually all cells of the body express class I MHC and contain the intracellular machinery required for generating peptides that bind to them. The histocompatibility leukocyte antigen (HLA)-A, B, and C heavy chains form the peptide-binding groove for human class I MHC and are associated with P2-microglobulin, a monomorphic light chain. Peptides bound This work was supported by Grants RO1 HD97002 and RO1 A126940 from the National Institutes of Health. From the Division of Immunology and Transplantation Biology, Stanford University School of Medicine, Stanford, California

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VOLUME 18 NUMBER 2 MAY 1998

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LEWIS

Figure 1. T-cell recognition of antigen and activation. The dp-T-cell receptor (TCR) recognizes antigen presented by the antigen-presenting cell (APC) in the form of antigenic peptides bound to MHC molecules on the APC surface. Most CD4 T cells recognize peptides bound to class II MHC, whereas most CD8 T cells recognize peptides bound to class I MHC. This MHC restriction is the result of a thymic selection process, and is caused by, in part, an intrinsic affinity of the CD4 and CD8 molecules for the class II and class I MHC molecules, respectively. Once antigen is recognized, the CD3 protein complex, which is invariably associated with the dp-T-cell receptor, transduces an intracellular signal that leads to T-cell activation. Engagement of the CD28 molecule on the T cell by B7-1 or B72 on the APC transduces an additional costimulatory signal that is essential for full Tcell activation.

to class I MHC molecules are typically only eight to nine amino acid residues in length and are mainly derived from proteins synthesized within the APC (Fig. 2). These peptides are usually fragments from normal self-proteins unless the cell’s cytoplasm contains foreign protein (e.g., as a result of infection with an intracellular pathogen, such as a virus). Class I1 MHC is mainly found on cells specialized for antigen presentation (e.g., dendritic cells, mononuclear phagocytes [Mq], B cells, and thymic epithelial cells), and present peptides mainly to CD4 a/P-T cells.127There are three types of class 11 MHC human molecules, DR, DP, and DQ, each consisting of an cx and a P chain that together form a peptide-binding groove (see Fig. 1). The peptides binding to these molecules typically range in size between 14 and 16 amino acids. Most class I1 MHC peptides are derived from phagocytosis or endocytosis of soluble or membrane-bound proteins (see Fig. 2).lZ7The invariant chain protein prevents occupation of the groove until the class I1 MHC molecule reaches a specialized endocytic compartment, in which peptide loading takes place. Release of the invariant chain and the loading with peptide is facilitated by the HLA-DM molecule (see Fig. 2). Class I1

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HC

de

ease(f arianl

Class I1 MHC Endocytic Compatfmeni

b

Golai

Figure 2. lntracellular pathways of antigen presentation. Foreign peptides that bind to class I MHC are derived predominantly from cytoplasmic proteins that are either synthesized de novo within the cell or that enter the cytoplasm, such as following fusion of a virus with the cell membrane. Peptide binding takes place within the endoplasmic reticulum. For class II MHC, the invariant chain prevents their binding peptides until they reach a specialized cellular compartment for class II MHC peptide loading. HLA-DM may facilitate removal of invariant chain in this compartment and the loading of peptides in this compartment. Once peptides derived from internalized proteins bind to class II MHC, the peptide/MHC complex is exported to the cell surface.

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MHC-associated peptides are self-peptides unless the cell internalizes foreign proteins derived from infection, transplantation, or immunization. dP-T CELL DEVELOPMENT

The development of lymphocytes of the T cell, B cell, and NK cells from less committed hematopoietic cells involves a common lymphocyte p r o g e n i t ~ rThis . ~ ~ progenitor gives rise to the prothymocyte, a lymphoid cell that lacks TCR-a or TCR-p chains. The prothymocyte expresses CD3 molecules in its cytoplasm and the CD7 protein on its surface, and has little or no expression of CD4 or CD8. CD7 is also found on mature T cells and NK cells suggesting a close relationship between these two cell types.’@Differentiation within the thymus is an obligatory step for most a/P-T cells and begins when the prothymocyte enters the subcapsular region of the thymus from the circulation (Fig. 3). Prothymocytes appear

Cell Type

Major Developmental Events

Prothymocyte

Migration into thymus from bone marrow or fetal liver

ImmatureThymocyte (CD4IoW CD8’OW)

Proliferation, TCR gene rearrangement

Subcapsular Region

.

v)

a

E Cortical Thyrnocyte ( ~ ~ 4 h i C~8high) Qh

Positive selection of the ap-TCR repertoire

Medullary Thymocyte (CD4”QhCD8’OWor CD4IoWCD8”gh)

Negative selection of the ap-TCR repertoire

Cortex

i= TCWCD

Emigration to periphery Peripheral CD4t and CD8+ T cells

CD4t T Cell

CD8+ T Cell

Figure 3. Putative stages of human thymocyte development. Prothymocytesfrom the bone marrow enter the thymus subcapsular region and give rise to progressively mature d p - T cell receptor (TCR) thymocytes, defined by their pattern of expression of the dp-TCWCD3 complex, CD4, and CD8. TCR-a and TCR-p chain genes are rearranged in the subcapsular region, positive selection occurs mainly in the thymic cortex, and negative selection occurs mainly in the medulla. Following these selection processes, medullary thymocytes emigrate into the circulation and colonize the peripheral lymphoid organs.

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to retain the ability to differentiate into the NK-lineage even after entering the thymus.148The thymus lacks self-replenishing lymphoid precursors or multipotent hematopoietic stem cellsIn and requires a continual input of prothymocytes for T-cell production. Thymocytes in the subcapsular region initiate TCR gene rearrangement. The TCR-p chain gene is always rearranged prior to the TCR-a chain gene. The unrearranged human TCR-P chain gene spans 685 kilobases of DNA on human chromosome 7 and consists of 46 potentially functional variable (V) gene segments located upstream of two constant (C) regions, each associated with one diversity (D) and six joining (J) segments (Fig. 4).IM The D segment first rearranges to a downstream J segment, with the deletion of intervening DNA, followed by rearrangement of a V segment to the DJ segment, resulting in a contiguous (VDJC) P chain gene segment. If this segment lacks premature translation stop codons, the TCR-P chain protein is expressed on the thymocyte surface in association with a pre-TCR-a chain protein and the CD3 signaling complex.1o3, This complex instructs the thymocyte to increase its surface expression of CD4 and CD8 and to rearrange the TCR-a chain gene. Rearrangement of the TCR-a chain gene then occurs, and involves the joining of V segments directly to J segments, without intervening D segments. The TCR-P/pre-TCR-a complex also signals the thymocyte to stop rearrangement of the other TCR-P chain allele, resulting in allelic exclusion, so that more than 99% of a/P-T cells express only a single type of TCR-P chain gene.'*O

T Cell Receptor P-Chain Gene

Immunoglobulin Heavy Chain Gene Rearrangement and isotype Switching

Rearrangement V&.N

DPI

J&*

C&

VHI~

DY.N

J

H

~

cc ~

c8

c%

c%

c.h cI cE

c%

Gerkline VDJ Rearrangement

Figure 4. Rearrangement of the human T-cell receptor-p chain gene. Rearrangement of dispersed V, D, and J gene segments is accomplished by deletion of intervening DNA, which allows expression of a full-length RNA transcript that can be translated into a functional T-cell receptor chain protein. A similar process is involved with rearrangement of the TCR-a, -y, and -6 chain genes, and with the heavy and light immunoglobulin chain genes. The heavy chain immunoglobulin gene can undergo isotype switching, in which the C region is changed without altering the antigen-combiningsite formed by the V, D, and J segments. lsotype switching from IgM to IgG1 is shown as an example.

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TCR diversity is generated by the largely random use of V, D, and

J segments in assembling the TCR-a and TCR-p chain genes.39The CDR3 region, in which the distal portion of the V segment joins either the (D)J segment, appears to be a particularly important source of a/P TCR diversity for peptide/MHC re~ognition.3~ Additional diversity results from imprecision in segment cleavage in preparation for recombination, and by random nucleotide addition to segment ends by the terminal deoxytransferase (TdT) enzyme. Thymocytes that have successfully rearranged and express a/PTCRs have a CD4I”WDSh1@ surface phenotype (see Fig. 3). Their a / p TCRs interact with self-peptide MHC complexes found on epithelial cells of the cortex of the thymus. The TCR interacts not only with the peptide in the groove but also in regions of the MHC that form the groove, for which it has an intrinsic affinity.*” If the TCR has sufficient affinity for peptide/MHC complexes, the thymocyte receives a signal allowing its survival (positive selection)? If this signal is absent or weak, the thymocyte dies by apoptosis, a process of cellular suicide that involves activation of caspases, a family of cysteine proteases. Positive selection is also influenced by interactions between MHC molecules and the CD4 and CD8 molecules. Class I MHC and class I1 MHC molecules have constant domains located outside of the peptidebinding groove that have affinity for CD8 and CD4, respectively. As a result of these interactions, most CD4QhCD81°w thymocytes (and their peripheral CD4 T-cell descendants) recognize peptides bound to class I1 MHC molecules, and most CD410wCD8h1gh T cells (and peripheral CD8 T cells) recognize peptides bound to class I MHC molecules (see Fig. 3). Positively selected CD4hLghCD810w and CD4lowCDShlph thymocytes enter the medulla and undergo a process called negative selection, in which they are eliminated by apoptosis if their TCR has too high an affinity for peptide/MHC complexes expressed on medullary dendritic cells.93 Negative selection helps eliminate a/P-T cells with TCRs that could pose a risk of autoimmune reactions, and is an important influence on the final TCR re~ert0ire.l~~ Positively selected thymocytes that are not eliminated by negative selection enter into the circulation as antigenically naive a/P-T cells and preferentially home to the peripheral lymphoid organs (see Fig. 3). Because the region forming the peptide-binding groove of MHC molecules is highly polymorphic in the human pop~lation,’~ a result of positive selection is that T cells have a strong preference for recognizing a particular foreign peptide bound to self MHC, rather than to the MHC of an unrelated individual. On the other hand, the fact that TCR has intrinsic affinity for MHC molecules accounts for the ability of an APC bearing foreign MHC molecules to activate a substantial proportion (up to several percent) of T cells (the allogeneic response). In the allogeneic response, T cells are activated by antigens that result from the combination of a foreign MHC with multiple self-peptides and that have not participated in the negative selection process in the medulla.

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Human Fetal Thymocyte Development and the T-cell Receptor Repertoire

Between 6 and 8 weeks of gestation the human fetal liver contains CD34+ lymphoid cells that appear to include prothymocytes, in that they can undergo differentiation into T-lineage cells in vitro under suitable conditions.131The human fetal thymus is first colonized with prothymocytes at approximately 8.5 weeks of gestation.75Shortly thereafter, thymocytes express proteins characteristic for T-lineage cells, including CD4, CD8, and the TCR-a and TCR-p chain proteins.75By 12 weeks of gestation, a clear architectural separation between the thymic cortex and medulla is a ~ p a r e n t 7and ~ Hassall’s corpuscles are evident shortly thereafter.61 Medullary dendritic cells in the fetus but not the adult express high levels of B7-l.ls1 B7-1 is a ligand of CD28,29a protein that is expressed by most fetal and postnatal thymocytes as well as most mature T cells. It is unclear if B7-1 plays a role in human fetal thymocyte development. By 14 weeks of gestation the three major human thymocyte subsets defined by surface expression of the CD4 and CD8 molecules-doublenegative (CD410WCD810w), double-positive (CD4hWD8hgh),and singlepositive (CD4h1@’CD8’ow or CD410wCD8h@’)-arefound in the subcapsular, cortical, and medullary regions, respectively, a pattern that persists in the postnatal thymus (see Fig. 3). At this time, CD4 and CD8 T cells are found in the fetal liver and spleen, and CD4 T cells are detectable in primary lymph node follicles: indicating the emigration of mature Tlineage cells from the thymus. Thymc cellularity increases dramatically during the last trimester and continues to do so postnatally, with peak thymus size reached at about 10 years of age. The CDR3 region of the TCR-P chain transcripts is reduced in length and sequence diversity in the human fetal thymus between 8 and 15 weeks of gestation, most likely due to decreased amounts of the TdT emyme.15* 58, 135 Because the CDR3 region of the TCR chains is a major determinant of antigen specifi~ity;~ such decreased CDR3 diversity theoretically could limit recognition of foreign antigens by the first-trimester fetal a / p TCR repertoire; however, any potential ”holes” in the a/P TCR repertoire of the human fetus from limitations in CDR3 are likely to be very subtle. This is suggested by the fact that the T-cell response to immunization and viral challenge is normal in mice that are completely deficient in TdT as a result of selective gene targeting.60By the second trimester, TdT activity and CDR3 length are both increased?8*135 and Vp and Va segment usage in the thymus and peripheral lymphoid organs is diverse and similar to that of adult T cells.“, lZ1, 137 T-CELL ACTIVATION AND EFFECTOR FUNCTIONS

Activation of peripheral a / p T cells by engagement of the TCR with foreign antigenic peptide bound to MHC leads to tyrosine phosphoryla-

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tion of the cytoplasmic domains of the CD3 complex.1o3The CD3 molecules, in turn, act as docking sites for molecules that propagate the activation signal by multiple These signals result in a stereotyped sequence of events that occur over days to weeks, including Tcell proliferation and differentiation into effector cells,'7o cytokine gene transcription,36and, in the case of cytotoxic T cells, the killing of adjacent target cells.9If the affinity of the TCR for peptide/MHC is weak, partial signaling may occur, leading to certain activation outcomes (e.g., cytokine production) but not others (e.g., cell proliferation)?' If the encounter of the T cell with antigenic peptide/MHC is relatively limited in terms of dose or of duration (e.g., following immunization with protein), the full activation program requires signals in addition to those provided by TCR engagement. These additional signals are In such cases of limited antigen collectively referred to as costimu2~tion.~~ exposure, engagement of the TCR without stimulation may not only fail to activate the T cell but, instead, render it anergic.'" Anergic T cells will not subsequently respond to antigen even when normally adequate costimulatory signals are provided by the APC. Anergy is an attractive model for the maintenance of tolerance by mature T cells to certain self antigens, particularly those that may not be expressed at sufficiently high levels in the thymus to induce negative selection. Major sources of costimulation are due to interactions between the B7 molecules, B7-1 (CD80) and B7-2 (CD86) on the APC and CD28 on the T cell?9 and CD40 on the APC with CD40-ligand, a TNF family cytokine, on the T cell (Fig. 5).M,178The CD28 molecule is constitutively expressed on most ol/P-T cells, whereas CD40-ligand is rapidly expressed within hours following TCR engagement by antigen.178 Antigenically naive CD4 and CD8 T cells, once activated, undergo clonal expansion and differentiation into effector T-cell populations (Fig. 6).170 Effector T cells are lymphoblasts in the active phases of the cell cycle (i.e., not Go), and have greater capacity for cytokine production and cell-mediated cytotoxicity and a reduced costimulatory requirement than antigenically naive T cells." Unlike antigenically naive T cells, effector T cells have high surface levels of high-affinity IL-2 receptors, the CD69 molecule, fas (a TNF receptor family member),*12and low 147 intracellular levels of bcl-2, a protein that protects against apopt~sis.~, Effector T cells tend to undergo apoptosis, particularly when fas is engaged by fas-ligand, a TNF family cytokine, unless antiapototic signals are provided by IL-2 or other cytokines, such as IL-6.4,175 Apoptosis is important for limiting the accumulation of effector T cells once they are no longer needed for the immune resp0nse.9~

CD4 T-cell Activation and Cytokine Secretion When antigenically naive CD4 a/P cells first encounter foreign peptide/MHC complexes (the primary immune response), they produce

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3

-

11-12

IL-12

Step 1

Step 2

Figure 5. T-cell-APC interactions early during the immune response to peptide antigens. Dendritic cells are probably the most important APC for antigenically naive T cells and constitutively express B7 and CD40 molecules on their cell surface. Engagement of the T cell by antigenic peptide bound to MHC on the dendritic cells, in conjunction with costimulation by B7/CD28 interactions, leads to T-cell activation (Step 1). The activated T cell expresses CD40-ligand on its surface, which engages CD40. This engagement activates the dendritic cells to produce cytokines such as IL-12 (Step 2). IL-12, in turn, promotes the differentiation of T cells into Thl-type effector cells that produce high levels of IFN y and low or undetectable amounts of IL-4.

a limited number of cytokines, inclyding IL-2 and CD40-ligand (see Figs. 5 and 6).170,178IL-2 is secreted and is an autocrine and paracrine growth factor of T cells, helping expand antigen-specific T cells. IL-2 also increases the capacity of effector T cells to produce additional cytokines upon their reactivation by antigen,170including IFN gamma (IFN-y), IL3, IL-4, and IL-5. CD40-ligand on the T cell engages the CD40 molecule ~ B on B cells, dendritic cells, and mononuclear phagocytes ( M V ) . ' ~For cells, CD40 engagement promotes their expression of antibody isotypes and their differentiation into a memory cell p0pu1ation.l~~ For dendritic cells and Mq, this engagement induces production of IL-12 and other cytokines (see Fig. 5).178IL-12, in turn, induces IFN-y secretion by NK cells, and, in conjunction with IFN-y, promotes the development of T helper 1 (Th-1) effector cells.51The Th-1-effector response, in which T cells produce IFN-y and IL-2 at high levels, and little or no IL-4 and IL5, is an important means of limiting infection by intracellular pathosuch as Toxoplasrna and Listeria. In cases in which antigenically naive T cells are activated by allergens or by helminth parasites and are not exposed to IL-12, they tend to develop into Th-2 T-cell effectors that produce high levels of IL-4, IL-5, and IL-13, and low levels of IL-2 and IFN-Y.~~ The Th-2 effector response is characterized by high levels of IgE production by B cells, IgE-dependent mast cell and basophil activation, and inflammation with eosinophils.

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CD4 T Cells

Resting Antigenically Naive Cell

CD8 T Cells

CD45

.1antigen on hc

tion by n on APC

Activationb

Activated Naive Cell

---. . -.

CD45RO

Primary Effector Cell

1

'\? \ \

I I

+ D

II

I

I

I I /

/

Resting Memory

Cell

11-2

Activated Memory Cell I

- 1

Secondary Effector Cell

11-2

11-2 gianules

Figure 6. Differentiation of antigenically naive and memory T cells into effector T cells. Antigenically n&ve CD4 or CD8 T cells are activated by antigen presented by APC and undergo expansion into a lymphoblast effector T-cell population. Cytokines produced by CD4 T cells, such as IL-2, may be important for the generation of CD4 and CD8 effector T cells. Effector T cells, when reactivated by antigen, have a greater capacity to produce multiple cytokines and to mediate cytotoxicity. Following initial activation, some T cells also persist as quiescent memory T cells. Activation of memory T cells by reexposure to antigen results in the generation of a secondary effector population. In the human, memory and primary and secondary effector T cells have high levels of surface expression of CD45RO compared with antigenically naive T cells, which mainly express CD45RA. CD45RA and CD45RO are isoforms of CD45, a cell surface protein-tyrosine phosphatase expressed on most hematopoietic cells.

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Activation and Differentiation of Cytotoxic T Cells a/P-T cells, particularly the CD8 subset, mediate antigen-specific, MHC-restricted cytolysis, an activity that is critical for resistance to viral infection. CD8 T cells when first activated by antigen are not effective killers, but, under the influence of cytokines such as IL-2, proliferate and differentiate into an effector T-cell lymphoblast population that efficiently kills (see Fig. 6).lmCD8 T-cell expansion and differentiation may be dependent on cytokines produced by CD4 T cells, such as IL-2, particularly if the amount of antigen exposure and costimulation is limited. Like CD4 effector T cells, most CD8 effector T cells have a relatively short life span and are eliminated by apoptosis following antigen clearance. In cases of human viral infection where class I MHC antigen presentation is markedly inhibited, such as with herpes simplex virus (HSV), class I1 MHC-restricted cytotoxic CD4 T cells may also play an important role in viral ~ l e a r a n c e . ~ ~ T cell-mediated cytotoxicity requires that the T cell binds to the target cell via multiple intercellular adhesion molecule interactions. Following adherence, if the TCR is engaged by antigenic peptide/MHC of the target cell, a killing program is executed in which the T cell secretes proteins (perforin and granzymes) and expresses fas-ligand on its surface.9 Perforin disrupts the target cell membrane and also allows the entry of granzyme molecules, which induce apoptosis by cleaving intracellular substrates of the target cell? Activated cytotoxic T cells also express fas-ligand, which induces apoptosis of target cells expressing the counterligand, fa^.^ Cytokines produced by effector T cells, such as IFN-y and TNF, may also directly inhibit intracellular viral replication in tissues, such as the liver, by a noncytotoxic m e ~ h a n i s m . ~ ~

Generation of Memory T Cells and Secondary Effector T Cells

After antigenically naive T cells are activated and expanded by antigen as part of the primary immune response, a small number of these cells persist as memory T cells (Fig. 6)j1,170 It has been estimated that the expansion of human memory T cells from antigenically naive T cells is the result, on average, of 14 cell divi~i0ns.l~~ Unlike effector T cells, memory T cells are not lymphoblasts and are in a GO-lphase of the cell cycle, rather than in an active phase (i.e., S or GJM). Memory T cells resemble effector T cells in that they have a reduced dependence on costimulation and a greater capacity to produce cytokines than antigenically naive T cells.17oMost human memory and effector CD4 and CD8 T cells can be identified by their surface expression of the CD45RO rather than the CD45RA isoform of CD45, a protein tyrosine phosphatase 193 About 40% of circulating adult CD4 T cells have this (see Fig. 6).138, CD45RA'owCD45ROh'ghsurface phenotype. Approximately 10% to 15% surface of circulating CD4 T cells have a CD45RAmediUmCD45ROmedium

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phenotype and memory- or effector-like but their relationship to CD45ROhighT celIs remains unclear. In contrast to memory or effector T cells, most antigenically naive T cells have a CD45RAhighCD45ROLow surface phenotype,’93although a minority of CD45RAh1ghT cells may be cells. revertants from either CD45RO’” or CD45ROmedium T cells in vitro Activation and propagation of CD45RAhighCD45R010w results in their acquisition of memory or effector cell-like features, including reduced costimulatory requirements, a CD45RA1OwCD45R0Qh phenotype, an enhanced ability to produce cytokines, such as IFN-.I and IL-4, and an increased ability to provide help for B-cell antibody production.34, This acquisition supports the notion that CD45RAhighT cells are precursors of CD45ROh’ph T cells, and that this differentiation occurs following T-cell activation. When memory T cells reencounter antigenic peptide or MHC complexes (recall antigen) as part of the secondary response, they are activated and undergo expansion and differentiation into a secondary effector population (see Fig. 6). The secondary immune response to recall antigen is typically more rapid and robust than the primary response to an antigen that has previously never been encountered because of the greater frequency antigen-specific T cells in the circulation and lymphoid tissue15o,193 as well as the enhanced function of these memory T cells and their secondary effector progeny.170Enhanced secondary responses by T cells can be observed months to decades after a single exposure to a new antigen, indicating that T-cell memory overall is table.^ Whether most memory T cells are long-lived or are continually generated in vivo remains controversial. Fetal and Neonatal T-cell Surface Phenotype

The percentage of T cells in the fetal or premature circulation gradually increases during the second and third trimesters of pregnancy through about 6 months of age,’58 followed by a gradual decline to adult levels during c h i l d h ~ o dThe . ~ ~ratio of CD4 to CD8 T cells in the circulation is high during fetal life (about 3.5) and gradually declines with age.7oThe levels of expression of the a/P-TCR, CD3, CD4, CD5, and CD8, and CD28 proteins on fetal and neonatal a/P T cells are similar to adult T cell^?^,^^, 142 Unlike adult antigenically naive T cells, most circulating fetal and neonatal T cells express high levels of the CD38 molecule.189 Because virtually all thymocytes are CD3Shigh,circulating fetal and neonatal T cells may represent an immature, transitional population. Circulating T cells in the term and preterm (22 to 30 weeks gestation) neonate and in the second trimester fetus predominantly express a CD45RAhighCD45RO’OW surface phenotype, characteristic of antigenically naive T cells in adults.E,125About 30% of circulating T cells of the term T cells, a population that is rare or neonate are CD45RALoWCD45ROLow Because these CD45RA’owCD45R010W T absent in the adult circ~lation.~~ cells are functionally similar to neonatal CD45RAkghCD45R010w T cells,

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and become CD45RAmidCD45RO'OW T cells when incubated in vitro with fibroblasts,14they appear to be immature recent thymic emigrants. These cells may undergo extra-thymic maturation to an antigenically naive (CD45RAhighcD45RO'ow) T-cell population over a period of days, as has been described in 10dents.l~~ Most studies have found that the healthy neonate and late gestation fetus lack circulating CD45ROl@' T cells, consistent with their limited exposure to foreign antigens. A postnatal precursor-product relationship between CD45RAhighCD45RAOLow and CD45RALowCD45ROhigh T cells is suggested by the fact that the proportion of a/P-T cells with a memory or effector phenotype, and the capacity of circulating T cells to produce cytokines, such as IFN-7 both gradually increase, whereas the proportion 70 These increases in the ability of antigenically naive T cells to produce cytokines and expression of the CD45ROhighphenotype are presumably due to cumulative antigenic exposure and T-cell activation. About 5% of circulating CD4 T cells in neonates, infants, and young children express high levels of CD45RA and IL-2 receptor OL chains and contain CD45RO transcripts.s4This T-cell subset may be a transitional population between antigenically naive and memory or effector T cells, although its antigen specificity has not been reported. Fetal T-cell Proliferation and Cytokine Responsiveness A minority but substantial proportion of T cells in the second trimester fetal spleen are CD45RALowCD45ROhigh, a T-cell population that is absent from the spleen of young infants.25These fetal CD45ROhighT cells express high levels of the IL-2 receptor alpha chain and proliferate with IL-2, suggesting that they have recently been activated and are undergoing expansion.2s In contrast to adult CD45ROhighT cells, these fetal spleen CD45ROhighT cells express low surface levels of CD2 and LFA-1 and proliferate poorly after activation with either anti-CD2 or anti-CD3 monoclonal antibodies, suggesting that they are not fully funct i ~ n a l Their . ~ ~ a/P-TCR repertoire is diverse, suggesting that these T cells are expanding in a non-antigen-specific manner. Such antigenindependent expansion can occur in cases in which the number of niches for T cells in the peripheral lymphoid tissue is large (e.g., following adoptive transfer of T cells into lymphopenic recipients), and it is possible that increased extrathymic niches for T cells may also apply to the rapidly growing fetus. However, it is unknown if these fetal spleen CD45RPgh T cells contribute to the postnatal T-cell compartment.

Neonatal T-cell Proliferation, Cytokine Responsiveness,and Cytokine Production

Most studies have found that circulating neonatal T cells and adult T cells have similar amounts of proliferation and IL-2 production in

response to the mitogenic lectins, bacterial superantigens, or to allogeneic 34, 76, 171, lS8Basal expression of the IL-2 receptor y chain is lower by neonatal T cells than by either adult CD45RAh1gh or CD45ROh'ph T cells.145However, activated neonatal T cells express high-affinity IL-2 receptors and proliferate in response to IL-2 as well or better than adult T cells,99,lS8 suggesting that these differences in basal IL-2 receptor expression are not functionally significant. Interestingly, in one report74neonatal mononuclear cells had decreased antigen-specific T-cell proliferation and IL-2 production in response to a protein neoantigen, keyhole limpet hemocyanin, compared with adult cells. These findings, which need confirmation, contrast with normal IL-2 production by neonatal T cells in response to alloantigen. The function of dendritic cells is reduced in the neonate80 (description follows), and these cells are critical for activation of antigenically naive T cells by antigen.'@Therefore, it is plausible that in the neonate, decreased dendritic function might compromise the presentation of soluble proteins to a greater degree than alloantigens, which are presented effectively by Mq as well as dendritic cells.37 In most studies, neonatal T cells and antigenically naive (CD45RAh@) adult T cells do not proliferate as well or produce as much IL-2 as unfractionated adult T cells after activation by anti-CD2 or anti-CD3 monoclonal antibodies.%,59, 163, 164 The production of most other T cellderived cytokines or expression of their cognate mRNAs by neonatal T cells has been reported to be either slightly (TNF)&or markedly reduced (IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-y and GM-CSF) compared with adult T cells using polyclonal stimuli for activation.30,43,47, 49, 95, Io1 However, adult antigenically naive T cells also have a reduced capacity to produce these cytokines compared with memory 'or effector T cells.s, 146 This reduced capacity suggests that, in most instances, the apparent cytokine deficiency of neonatal T cells can be accounted for by their antigenically naive status, rather than developmental immaturity. Reduced IL-4 and IFN-y mRNA expression by neonatal T cells is primarily due to reduced transcription of these cytokine genes.lol The IFN-y gene's DNA is methylated to a greater degree in neonatal and adult CD45RAh1ghCD45ROow T cells than in adult CD45RALowCD45ROh1gh T cells, potentially decreasing the accessibility of this gene to transcriptional activator proteins.lo6In the case of decreased IL-3 production by neonatal T cells, reduced IL-3 mRNA stability rather than decreased gene transcription may be the major me~hanisrn.~~ As with antigenically naive adult T cells, polyclonal activation of neonatal T cells result in their acquisition of the characteristics of effector T cells. These acquired characteristics include a CD45RAIowCD45ROhlgh surface phenotype, an enhanced ability to be activated by anti-CD2 or anti-CD3 monoclonal antibodies, and an increased capacity to produce cytokines (e.g., IL-4 and IFN-y)?4,47, 76, 130 Again, this capacity favors the notion that neonatal T cells are not intrinsically deficient in the capacity to produce cytokines, but instead, lack antigenic experience. loor

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Fetal and Neonatal T-cell Expression of CD4O-Ligand

The pattern of expression of CD40-ligand by fetal and neonatal T cells is distinct from all other T cell-derived cytokines reported to date: In one a substantial proportion of circulating fetal T cells between 19 and 31 weeks of gestation expressed CD40-ligand in vitro in response to polyclonal activation. In contrast, T cells from later gestational age fetuses and from neonates do not have this capacity to produce CD40-ligand.2°,45, %, 117 Whether fetal T cells that can express CD40ligand have a distinct surface phenotype from those lacking this capacity is not known. In most studies, activated neonatal T cells express markedly lower amounts of CD40-ligand surface protein and mRNA than either adult CD45RAfiphCD45RO'owor CD45FL4loWCD45ROh'gh CD4 T ~ells.4~. %, 117 Thus decreased CD40-ligand expression is not accounted for by the lack of a memory or effector population in the neonate T-cell compartment and appears to represent a true developmental limitation in cytokine production. As for most other T cell-derived cytokines, when neonatal T cells are activated in vitro into an effector T-cell population, they acquire a markedly increased capacity to produce CD40-ligand, demonstrating that this reduction in cytokine expression is not a fixed phenotype.&,117 Given the importance of CD40-ligand in multiple aspects of the immune response,'78 limitations in CD40-ligand production could contribute to decreased antigen-specific immunity mediated by Th-1 effector cells and B cells in the neonate. However, all of the studies that found a relative deficiency of CD40-ligand expression by neonatal T cells used calcium ionophore and phorbol ester stimulation, a combination of pharmacologic agents that maximizes the production of most cytokines but may not accurately mimic physiologic T-cell activation. Moreover, a recent report has found similar levels of CD40-L surface expression by neonatal and adult T cells in response to activation by anti-CD3 monoclonal antibody.166Given these conflicting results, it will be important to assess whether expression of CD40-ligand is reduced during antigenspecific T-cell activation during the neonatal period and early infancy. Some CD40-ligand can be expressed by neonatal T cells in response to allogeneic stimulation in vitro and can induce IL-12 production by dendritic cells,118but it is unclear if such production is equivalent to that by adult T cells in this context. Costimulation of Neonatal T Cells

Neonatal T cells produce IL-2 and proliferate as well as adult T cells in response to mouse cells expressing human B7-1 or B7-2 and anti-CD3 monoclonal antibody, indicating that CD28-mediated signaling is Anti-CD28 mAb treatment of neonatal T cells also markedly augments their ability to produce IL-2 and proliferate in response to anti-CD2 monoclonal antibody ~timulation.~~ However, there is one report that

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neonatal T cells differ from adult CD45RAh'phcD45RO'owT cells in their tendency to become anergic rather than competent for increased cytokine secretion following priming with bacterial superantigen bound to class I1 MHC-transfected murine fibrobla~ts.'~~ Superantigens activate T cells by binding to a portion of the TCR-p chain outside of the peptidebinding groove. These results, which need confirmation, suggest that neonatal, and presumably, fetal T cells, have a greater tendency than antigenically naive adult T cells to become anergic under conditions in which costimulation (e.g., via B7 or CD40 on the APC) may be limiting. Th-1 and Th-2 Effector Generation

Neonatal T cells express functional surface receptors for IL-4 and IL-12, including the activation-induced IL-12 p2 subunit'41because activated neonatal T cells can also be differentiated in vitro into either Th1- or Th-2-like effector cells with the addition of IL-12 or IL-4, respect i ~ e l y .14', ~ ~ , Interestingly, purified CD4 CD45RAhighT cells from neonates have been reported to proliferate substantially more in response to IL-4 than these cells from suggesting a mechanism by which neonatal T cells might be more prone to become Th-2 effectors than antigenically naive adult T cells. Fetal and Neonatal T Cell-Mediated Cytotoxicity

The recent and growing use of cord blood for bone marrow transplantation has made it of practical interest whether neonatal T cells are capable of mediating cytotoxicity and potential graft rejection. Neonatal T cells are less effective than adult T cells as cytotoxic effector cells, following their generation in vitro by incubation with allogeneic targets.", I4O These studies have not directly combared neonatal T cells with purified adult antigenically naive T cells, however, and results using unfractionated T cells may be skewed towards demonstrating greater activity with the adult samples because memory or effector T cells may be more readily primed for cytotoxicity?, Neonatal CD8 T cells have also been reported to lack constitutive expression of perforin, whereas approximately 30% of adult CD8 T cells contain this protein.l0 Whether these differences are accounted for by the lack of memory or effector CD8 T cells in the neonate is unclear. Fetal and Neonatal T-cell Responses to Infection, Allografts, and Blood Transfusion

Congenital infection with viruses or Toxoplasma during the second and third trimesters may result in the appearance of CD45RW&T cells in the circulation and an inversion in the ratio of CD4 to CD8 T cells,Z1* 78,

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which suggests that fetal CD8 T cells can be activated and expanded in vivo in response to serious infection. These alterations may also be present in the circulation at birth as well as through early infancy,7l. although their sensitivity and specificity for diagnosing congenital infection remains unclear. It is also uncertain whether these memory or effector-like T cells are functionally competent. Pathogen-specific T-cell proliferative responses and cytokine responses (IL-2 and IFN-y) of infants and children who have been congenitally infected (e.g., with syphilis, CMV, VZV, Toxoplasma), are often markedly lower or absent than in those with postnatally acquired infection,”, 53* lo5,lZ3,n4*167 which is particularly evident when these infections occur in the first or second trimester. For severe infections occurring during the first trimester, a direct deleterious effect on T-cell development is a possible mechanism. However, T cells from infants and children with congenital toxoplasmosis retain the ability to respond to alloantigen, mitogen, and, in at least one case, tetanus toxoid.lo5This ability suggests that these reduced pathogen-specific responses may be more often due to mechanisms that result in antigen-specific unresponsiveness (e.g., antigen-specific anergy, deletion, or ignorance [the failure of the T cell to be initially activated by antigen]).lgOAs discussed previously, it is unlikely that a decreased TCR repertoire limits these immune responses, particularly in cases occurring after the first trimester. Decreased responses may not apply to all congenital pathogens because in one study, most 10-year-old children who were congenitally infected with mumps had delayed-type hypersensitivity reactions to mumps antigen, indicating the persistence of functional mumps-specific memory T cells.’ Postnatal infection of neonates with HSV results in antigen-specific proliferation and cytokine production (IL-2 and IFN-y) production by CD4 T cells; however, these responses are decreased and delayed in 169 their appearance compared with adults with primary HSV infe~tion.2~, Infants between 6 and 12 months of age also have moderately lower IL2 production in response to tetanus toxoid than older children and Taken together, these factors suggest that either antigen-specific memory CD4 T-cell generation or function is decreased during early infancy, particularly soon after infection or immunization. Whether this reflects limitations in antigen processing or in T-cell activation and costimulation, proliferation, and differentiation remains unclear. There have been few studies of antigen-specific cytotoxic T-cell responses in the fetus. In one case of congenital HIV-1 infection, HIVspecific cytotoxic T cells were detected at birth, which indicates that the ability of the fetal T cells to be activated by viral antigen and undergo expansion is at least partially intact.’” Studies of cytotoxic responses to HIV in perinatally infected infants suggest that the cytotoxic response may be reduced and delayed in appearance compared with adults with recent infection.lZ8Whether such delayed appearance and decreased responses of cytotoxic effector T cells in the neonate and, presumably the fetus, applies to other pathogens remains to be determined. Obviously, in

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the case of HIV, it is possible that these decreased responses are accounted for by HrV-1 having a greater suppressive effect on immune function in the young infant than in the adult. Experiments using human severe-combined immunodeficiency (SCID) mouse chimeras also suggests that second trimester human fetal T cells are capable of becoming cytotoxic effector T cells in response to foreign antigens and in rejecting solid tissue al10grafts.l~~ Persistence of donor lymphocytes and of graft-versus-host disease (GVH) is a rare complication of intrauterine transfusion in the last trimester, as well as in postnatal transfusion of premature neonates when unirradiated cells are used. This complication suggests that T-cell rejection mechanisms are competent in most of these recipients, although it may be that the risk of engraftment is higher in the fetus and neonate than in older individuals. A recent study has found that a T-cell response to alloantigens can be detected in newborns following in utero irradiated red blood cell transfusions from unrelated donors1s3and that these neonates have a significantly greater percentage of CD45ROhighT cells than healthy These findings support the notion that fetal T cells have the capacity to mediate allogeneic responses in vivo, although it is unclear if these responses are equivalent to those that would occur postnatally with mismatched red blood cell transfusion.

APC FUNCTION

By 12 weeks of gestation, the expression of class I and class I1 MHC molecules by a variety of fetal tissues is evident,77,119 and all of the major "professional" antigen-presenting cells (macrophages, B cells, Langerhans' cells, dendritic cells) are present. Class I MHC expression by neonatal lymphocytes has been reported to be lower than by adult cells,S5 although this difference needs confifmation and may not be functionally significant. Class TI MHC molecule expression by fetal APC in tissues appears to be similar to that of the adult, and expression by neonatal monocytes and B cells is either similar to or greater than that by adult cells.85,142 Fetal tissues are also vigorously rejected after transplantation into non-MHC-matched hosts, indicating that the level of surface MHC expression on fetal tissue, particularly of class I MHC, is sufficient to initiate a vigorous allogeneic response by host cytotoxic T cells. Class I1 MHC-mediated antigen presentation also appears to be grossly intact because neonatal and adult monocytes are similarly effective in presenting soluble protein antigens or alloantigens to induce T proliferation,32,142 a response that is mainly class I1 MHC-dependent. However, these results do not exclude the possibility of more subtle deficiencies in antigen presentation in the fetus and neonate, particularly under conditions that more stringently test APC function (e.g., during infection with herpesviruses that inhibit peptide loading of MHC by multiple mechanisms).

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As discussed previously, both antigen-specific T-cell responses and T cell-dependent antibody production by the fetus and neonate in response to infection or immunization tends to be reduced compared with that in older individuals. This reduction potentially could occur because of limitations in APC function as well as in intrinsic immaturity in Tcell function. Dendritic cells are typically the most important APC for presentation of protein antigens to antigenically naive T cells.194After dendritic cells encounter antigens in tissue and process them, they move to the lymph nodes and spleen, where they present these antigens to T cells. Dendritic cells express high levels of adhesion molecules and costimulatory molecules that may facilitate T-cell activation and also express cytokines, such as IL-12, in response to CD40 engagement that may be particularly important for the development of Th-1 effector responses.178,194 More recently, it has been appreciated that activated dendritic cells can produce CD40-ligand and may promote antibody production by B cells independently of T cells, at least in ~ i t r oThus, .~~ limitations in dendritic cell function might reduce antibody responses via an indirect effect on T cells, as well as a direct effect on B cells. Primary dendritic cells of the neonatal circulation are substantially less effective than analogous adult cells in enhancing lectin-induced Tcell proliferation or for inducing allogeneic responses by either adult or neonatal T cells.8oThis decreased activity is associated with substantially reduced levels of class I1 MHC DR molecules and the adhesion molecule, ICAM-1.80 If decreased function also applies to neonatal dendritic cells in tissues, this could significantly delay or limit the T-cell response to newly encountered protein antigens. Whether neonatal dendritic cells express reduced amounts of IL-12, CD40, and B7 costimulatory molecules remains to be determined. In addition to antigen presentation, the APC may also serve to orchestrate the immune response by producing proinflammatory cytokines, such as TNF, and chemotactic cytokines, such as a- and P-chemokines. Neonatal mononuclear cells enriched in monocytes produce substantially less TNF than adult cells in response to Listeriu, particularly during the first few hours of incubation,156and, if such reduced production occurs in vivo, this could limit the early host response to intracellular pathogens. Monocytes from premature infants (23-32 weeks gestation) also produce less IL-8, an a-chemokine, in response to TNF treatment than either adult or term neonatal m o n ~ c y t e sWhether . ~ ~ ~ fetal or neonatal monocytes are limited in their capacity to produce other chemokines, of which more than 30 have been identified in humans, remains to be determined. $6-T CELLS

T cells expressing y/S-TCR are rarer than a/P T cells in the circulation and most human tissues, with the exception of mucosa, such as the intestinal epithelium. Although some y/S-T cells can recognize conven-

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tional peptide antigens presented by MHC, this is probably not true for the majority of them.31For example, recognition of the HSV-encoded gI glycoprotein by murine y/6-T cells is direct and does not require antigen processing. Some human y /S T-cell clones recognize small phosphatecontaining nonpeptide molecules, similar to those expressed by Mycobacteria.31In most cases, however, the identity of the antigens recognized by y/6-T cells is unknown. Experiments with mice genetically deficient in y/S-T cells or in which these cells are depleted by antibody treatment suggest that they contribute to defense of the host against intracellular pathogens, including HSV,155which is particularly evident when a/p-T cell function is compromised. Like cytolytic ct/P-T cells, activated y/6T cells (e.g., with anti-CD3 monoclonal antibody) express perforins and granzymes, mediate cytotoxicity against tumor cells and other cell targets, and secrete cytokines, such as IFN-y and TNF. Rearrangement of the human y- and 6-TCR genes begins shortly after colonization of the thymus with lymphoid cells during fetal gestation and TCR-S protein expression is detectable by 9.5 weeks of gestation." Differentiation of y / 6 T cells may occur by a pathway that is largely or completely independent of that for a/P thymocytes." Whether y / 6 T cells undergo positive and negative selection similarly to a/pthymocytes remains controversial.3l y /S T cells comprise about 10% of the circulating T-cell compartment at 16 weeks, a percentage that gradually declines to less than 3% by term.114,125 Although there is potential for the formation of a highly diverse y/G-TCR repertoire, peripheral y/S T cells use only a small number of V segments, which vary with age and with tissue location. Most y/6 thymocytes of the first trimester express V62 segments, followed by thymocytes that express V61, which predominate at least through infancy. Most circulating fetal and neonatal y/S T cells are VylV61. By 6 months of age, T cells bearing Vy2V62 segments become predominant and remain so during adulthood,lZ2most likely due to their preferential expansion in response to an ubiquitous antigen or antigens. In contrast to the fetal thymus and circulation, V62 T cells predominate in the fetal I9l and appear prior liver and spleen early during the second to y/6-thymo~ytes,7~, ll1 suggesting that they be produced extrathymically by the fetal liver. Neonatal y/6-T cells express lower levels of serine esterases than adult y/6-T cells, suggesting they may be less effective as cytotoxic cells.159y/S-T cell clones derived from cord blood also have a markedly reduced capacity to mediate cytotoxicity against tumor cells and respond weakly to Mycobacterium extracts than y/S-T cell clones derived from adult peripheral blood.114Because these neonatal clones also had lower CD45RO surface expression than the adult clones, their reduced activity may reflect their relative antigenic naivete compared with most adult y / 6-T cells. In contrast to neonatal a/p-T cells, however, activation and propagation of these cells in culture does not appear to enhance their function relative to the adult y/G-T-cell population. The function of fetal liver y/6-T cells remains unclear. A single report suggests that they are

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enriched in cytotoxic reactivity against noninherited maternal class I MHC,'l' and could help prevent the engraftment of maternal T cells into the fetus. B CELLS AND IMMUNOGLOBULINS

Mature B cells have cell surface immunoglobulins (synonymous with antibody) composed of two identical heavy chains and two identical light chains linked by disulfide bonds. B cells are activated to proliferate and differentiate into immunoglobulin-secreting cells after their surface immunoglobulin (sIg) binds antigen. These antigens are typically intact proteins or other molecules, such as complex carbohydrates, and their recognition by immunoglobulin is usually highly sensitive to alterations in their three-dimensional structure. The sIg molecule is invariably associated with a group of proteins, Ig-a,-p, and -y, that transmit signals to the cell interior following sIg engagement.139Full B-cell activation by protein antigens usually requires help from activated T cells in the form of soluble and cell-surface cytokines, and is also necessary for immunoglobulin isotype switching. T-cell help may be dispensable for B-cell activation if antigen has multiple repeating subunits (e.g., complex polysaccharides) and can cross-link multiple sIgs of a B ce11.160 Analogous to the TCR, the amino terminal portion of the Ig chains is highly variable as a consequence of the assembly of V, D, and J gene segments to a monomorphic C region (see Fig. 4).39,139 The heavy immunoglobulin chain consists of V, D, J and the C region segments. The light chain is similar, except that it lacks D segments. Immunoglobulin heavy chain gene rearrangement occurs at the pro-B-cell stage, and involves sequential D to J and then V to D segment joining.'39 If this joining is productive, the heavy chain pairs with a surrogate light chain on the cell surface, directing the pre-B cell to stop rearrangement of the other heavy chain gene allele, and to start light chain gene rearrangement. This results in the mature antigenically naive virgin B cell usually expressing only a single type of immunoglobulin heavy chain, whereas allelic exclusion of the light chain is less efficient. Pro-B cells and pre-B cells are found only in the bone marrow, fetal liver, and fetal omenhun. The rearrangement process maximizes the generation of diversity at the expense of precision, and, consequently, most pre-B cells fail to produce a functional immunoglobulin molecule and die. As for TCR chains, immunoglobulin diversity is generated by the various combinations of V, D, and J segments joined together, and the imprecision in the joining process from random nucleotide loss or TdT-mediated nucleotide addition at the junctions. As with the TCR, the CDR3 region is particularly important in the generation of antigen recognition diversity by immunoglobulin. As discussed below, further diversification of immunoglobulin specificity is possible later in B-cell differentiation by a process known as somatic m ~ t a t i 0 n . I ~ ~

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There are two types of light chains, kappa or lambda, each containing a distinct C region. The C region of the heavy chain defines immunoglobulin isotype or isotype subclass, of which there are nine (IgM, IgD, IgG1, IgG2, IgG3, 1 6 4 , IgA1, IgA2 and IgE) (see Fig. 4). Antigenically naive B cells express sIg of the IgM and IgD isotypes, with IgD generated by alternative RNA splicing of the heavy chain transcript. All other immunoglobulin isotypes or subclasses are generated by isotype switching, in which a portion of the C region of the heavy chain gene is replaced with another isotype-specific segment, but the aminoterminal antigen combining site is preserved (see Fig. 4).139The C region also determines whether antibodies fix complement and bind to Fc receptors on leukocytes and to those involved in transplacental transport to the fetus. B-Cell Maturation, Selection, and Clonal Expansion

B cells, like T cells, appear to undergo positive and negative selection prior to their activation by foreign antigens. Evidence for both of these selective processes in humans comes from an analysis of the Bcell immunoglobulin re~ert0ire.l~ Although positive selection of B cells remains a poorly defined process, it is clear that negative selection can eliminate (clonal deletion) or inactivate (clonal anergy) potential autoreactive cells.63Mature antigenically naive B cells leave the bone marrow and colonize the peripheral lymphoid organs. B-cell activation and proliferation occurs when their sIg is engaged by antigen under appropriate conditions, such as in areas of the lymph node and spleen enriched in T cells and follicular dendritic cells. The follicular dendritic Activation cell is an APC specialized for antigen presentation to B results in the clonal expansion of B cells capable of secreting immunoglobulin to this antigen; the more avidly the given cell binds to the antigen, the stronger the stimulus is to proliferate. During the initial immune response, most antigenically naive B cells express antibodies with relatively low affinity for antigen. Antigen-specific B cells migrate to the primary follicles of the peripheral lymphatic tissue in small numbers, where they proliferate strongly, leading to the formation of germinal centers.86 Within the germinal centers, immunoglobulin with higher-affinity antigen-combining regions are generated by somatic mutation, the accumulation of random point mutations within the CDR3 region of existing V, D, and J Somatic mutation can occur independently of isotype switching in human B cells. For example, somatic mutation of the immunoglobulin heavy chain is common in adult B cells that express surface IgM but not IgD or other isotypes; these cells appear to represent a population of memory B cells that have not undergone isotype switchingB8B cells that have somatically mutated sIg with an increased affinity for antigen proliferate more effectively and will tend to predominate. B cells with high affinity immunoglobulin leave the germinal center to

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persist as memory B cells or immunoglobulin-secreting plasma cells. The encounter of B cells with CD23 and soluble cytokines provided by follicular dendritic cells may promote plasma cell differentiation, whereas encounter with CD4O-ligand may favor B-cell differentiation into memory cells.178Memory B cells can persist in a nonmitotic state in lymphoid organs for at least 6 months.152 Regulation of Immunoglobulin lsotype and Class Switching by Cytokines and Immunoglobulin Secretion

Cytokines derived from T cells or other cell types play an important role in promoting or inhibiting switching to a specific isotype. For protein antigens, isotype switching critically depends on expression of CD40-ligand by activated T ~ e l 1 s . Engagement l~~ of CD40 on the B cell in conjunction with other signals provided by cytokines, such as IL-4 and IL-10, markedly enhances immunoglobulin production in vitro and promotes class-switching.178 Cytokines also have important selective influences on isotype switching (e.g., IL-4 promotes switching to IgE and IgG4). During the differentiation of B cells into memory cells or plasma cells, most switch to a single isotype by gene rearrangement. Sequential isotype switching can also occur. B-cell maturation into plasma cells results in alternative RNA splicing of the immunoglobulin molecule so that it is secreted rather than embedded in the cell membrane. Maturation of B cells into plasma cells is associated with a marked increase in their capacity to secrete immunoglobulin and loss of surface Ig expression. Plasma cells rather than mature B cells account for the bulk of secreted immunoglobulin. Plasma cells are concentrated in peripheral lymphoid tissue, liver, and bone marrow, as well as in lymphoid tissue of the gastrointestinal and respiratory tracts. Ontogeny of B Cells and Immunoglobulin

Pre-B cells are first detected in human fetal liver and omentum by 8 weeks of gestation and in fetal bone marrow by 13 weeks of gestation.57, After 30 weeks of gestation, pre-B cells are found only in bone marrow. At mid-gestation, marrow pre-B cells in which surface IgM is coexpressed on the cell surface with surrogate light chains are evident.l16 B cells expressing surface IgM are present by 10 weeks of gestation.57Fetal B cells at this stage express IgM without IgD, unlike surface IgM-positive adult B cells found in the peripheral lymphoid organs, most of which also express surface IgD.57,66 Experiments in mice have shown that exposure of IgM + IgD - B cells to antigens functionally inactivates them (clonal anergy),'@which raises the possibility that antigen exposure in utero may tend to induce specific B-cell tolerance

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rather than an antibody response. This tolerance may account for the observation that early congenital infection, such as with mumps, can result in pathogen-specific defects in immunoglobulin production despite normal T-cell responses, such as delayed type hypersensitivity.’ For immunoglobulin production that requires T-cell help, such antigenspecific defects could also reflect the induction of T-cell tolerance. Between 10 and 12 weeks of gestation, B cells bearing sIg of the IgA, IgG, and IgD isotypes appear. The frequency of B cells in tissues then rapidly increases so that by 22 weeks gestation, the proportion of B cells in the spleen, blood, and bone marrow is similar to that in the adult.57,66 In contrast to adult B cells, which express IgM plus IgD or IgG or IgA alone, neonatal B cells have been reported to express IgG or IgA with IgM plus IgD.57This result, which was based on fluorescent microscopy, needs confirmation by multiparameter flow cytometry. True germinal centers in the spleen and lymph nodes are absent during fetal life but appear the first months after birth, presumably as a result of postnatal antigenic stimulation.86 CDlO is expressed by pre-B cells, and in the adult is lost with their differentiation into mature B cells. In contrast, most fetal bone marrow and spleen B cells express CD10?4,133 Although this expression raises the possibility that these cells might be a transitional population between pre-B cells and fully mature B cells, there is no evidence that these CDlO+ B cells are functionally immature based on their ability to undergo isotype switching (description follows).733 Another distinct feature of fetal B cells is their high frequency of CD5.733More than 40% of B cells in the fetal spleen, omentum, and circulation at mid-gestation are CD5 + 17, I6l but lesser numbers are found in the fetal liver and bone m a ~ r o w The . ~ preponderance of CD5+ B cells noted in the fetus is also observed in the neonatal c i r c ~ l a t i o nand ~ ~ gradually declines with postnatal age. Most of these CD5 + B cells appear to repr,esent a lineage separate from CD5- B cells, termed B1, a notion supported by the fact that these two populations are subject to different positive and negative influences of the preimmune (i.e., prior to exposure to foreign antigen) immunoglobulin repertoire.18 B1 cells, including those in the fetus and newborn, have a greater tendency to produce autoantibodies that are directed against self-antigens, such as DNA, than 82 cells.”, 87 B1 cells have been proposed to play a role in regulation and development of the immune system in early ontogeny, perhaps in the induction of tolerance to self-antigens. The Immunoglobulin Repertoire in the Fetus and Neonate

In the early- to mid-gestation human fetus, immunoglobulin heavy chain V segment usage is restricted compared with that in the The V segments that are expressed are scattered throughout the heavy ~ , ~ their ~~ CDR3 length is shorter than at birth, chain gene ~ O C U S , ’ ~but

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presumably because of decreased TdT The B-cell repertoire increases during gestation.38By birth it is highly diverse in the use of heavy chain V segments and has CDR3 regions that are similar in length to adult B cells for IgM and IgA, but not for IgG transcript^."^ Some limitations in V segment repertoire may persist through the neonatal period, and even into infancy.177 However, there is considerable flexibility by the immune system for generating immunoglobulin diversity (e.g., mice containing a single V heavy chain gene segment have normal immunoglobulin responses) (personal communication, MM Davis, 1998). Therefore, it remains unclear if these developmental restrictions in V segment usage or CDR3 diversity significantly limit the ability of the fetus or neonate to produce specific antibodies. The finding of frequent somatic mutations in IgG and IgA transcripts in the neonatal B cells indicates that somatic mutation is operative by birth.115

Fetal and Neonatal T Cell-Dependent Immunoglobulin Production and lsotype Switching IgM, IgG1, IgG2, IgG3, IgG4, and IgE production by neonatal B cells and adult antigenically naive B cells are similar using activation conditions that mimic T cell-derived CD40-ligand and soluble cytokine signals (e.g., CD40-ligand expressing fibroblasts in combination with IL4 or IL-10 or with cytokine-containing supernatants from activated T cells).19,157 Isotype switching has also been obtained with fetal B cells, including those expressing CD10, and pre-B cells from as early as 12 weeks of ge~tati0n.l~”’~~ Neonatal B cells produce lower amounts of IgG than adult B cells when these cells are cultured with adult T cells and a However, polyclonal T-cell activator, anti-CD3 monoclonal antibody.la, these differences may be due to the fact that the adult B-cell population contains memory B cells, most of which have already undergone isotype switching, although these cells are largely absent from the neonate. Experiments in which human B and T cells either develop in or are adoptively transferred into SCID mice also suggest that fetal and neonatal B cells are capable of isotype switching and immunoglobulin production when appropriate T cell-derived signals are present.26,176, lSo When neonatal T cells are activated for only hours and then fixed, they provide substantially less help for B cell immunoglobulin production and isotype switching by antigenically naive B cells than do similarly activated and fixed adult T ~ e 1 l s . lThe ~ ~ major source of help provided by such fixed T cells is likely to be mediated by CD40-ligand. Taken together, these results indicate that neonatal B cells have a capacity similar to adult B cells for activation and isotype switching in response to CD40 engagement and soluble cytokines. Limitations in fetal and neonatal B-cell immune responses largely may be due to reduced production of T cell-derived cytokines, particularly CD40-ligand. It is also plausible that limitations in neonatal dendritic cell function may limit B

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cell responses, both by a direct mechanism and an indirect mechanism involving T cells. Immunoglobulin-secreting plasma cells are detectable by week 15 of gestation, and those secreting IgG and IgA are first observed at weeks 20 and 30, IgM and IgG synthesis has first been detected as early as 12 weeks in fetal organ cultures.62In general, neonatal B cells can differentiate into IgM-secreting plasma cells as efficiently as adult cells, and, as discussed previously, can undergo isotype switching effectively when their CD40 molecule is engaged. One study has found that T cell-dependent immunoglobulin production by neonatal CD5 + and CD5 - B cells is more readily inhibited by agents that raise intracellular CAMP,such as prostaglandin E2, than are adult B cells.165It is unclear if adult antigenically naive B cells are resistant to these inhibitory effects mediated by CAMP. T Cell-Dependent Antibody Responses to Vaccines and Congenital Infection

In cases in which congenital infection is severe and occurs during the first or second trimester, detectable specific immunoglobulin production may not occur by birth or, in some cases, even late into c h i l d h o ~ d . ' ~ ~ This unresponsiveness appears to reflect a lack of T-cell help, at least in part, because antigen-specific T-cell proliferative responses are also frequently reduced, as discussed previously. However, congenital infection with Toxoplasma frequently results in circulating IgE and IgA antiToxoplasma protein antibodies at birth, indicating that T cell-dependent isotype switching and immunoglobulin production can occur during fetal life, at least for certain pathogens.*29These occurrences suggest that if antigen tends to produce B-cell tolerance in the human fetus, the induction of tolerance is not absolute in this context. The antibody response to hepatitis B prqtein vaccine, a T celldependent antigen, is consistently lower in the neonate and young infant than in older children and adolescents.1s6For several protein or proteinconjugate vaccines, preterm infants have reduced antibody responses compared with term infants of the same chronologic age. Given the normal responses of neonatal and fetal B cells in vitro, these results suggest that the function of T cells or of non-B cell APCs that participate in the antibody response may be decreased in the young infant, particularly the premature (e.g., with inactivated poliovirus vaccine).2 Given the recent finding of substantially reduced production of CD40-ligand by neonatal T cells, it will be of interest to determine whether production of this cytokine by antigen-specific T cells expanded by vaccination is also reduced in the young infant, particularly the premature. NK CELLS

Natural killer (NK) cells are large granular lymphocytes identified by their lack of TCR or Ig gene rearrangement and their surface expres-

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sion of CD16, a Fc receptor for antibody, of CD56.91Almost all adult NK cells are CD16 + CD56 and about 50% express CD57.126NK cells are mainly produced in the bone marrow and appear to be derived from a common T and NK cell precursor cell, possibly the prothymocyte. The close relationship of NK cells to T lymphocytes is suggested by the transient cytoplasmic expression of the CD3-epsilon (E) and -6 components by unactivated fetal NK cells.126All NK cells express the CD3-zeta (5) chain, which signals via an association with CD16 an Fc receptor for antibody. NK cells are functionally defined by their ability to lyse virally infected or tumor-target cells in a non-MHC-restricted manner that does not require prior sensitization. NK cells recognize self-class I MHC expressed on normal cells by killer inhibitory receptors (KIRs), and engagement of the KIR delivers an inhibitory signal to the NK cell preventing cytoto~icity.~~ The KIR is not engaged when self-MHC expression is reduced or, possibly, if portions of the MHC are masked by certain peptides. Triggering of NK cell-mediated cytotoxicity appears to result from a combination of the lack of KIR-mediated inhibitory signal in conjunction with activation signals provided when ligands on the target cell engage molecules on the NK cell, such as CD2 and LFA-1.91 For example, the ability of NK cells to lyse herpesvirus-infected cells is accounted for, in part, by the low levels of class I MHC expression by the target cell, due to herpesvirus-mediated intracellular inhibition of class I MHC antigen presentation. The importance of NK cells in the control of herpesvirus infections in humans is illustrated by their severity in rare patients with the selective absence of NK cells.12,98 The cytotoxic activity of NK cells is also triggered by engagement of antigenIgG complexes through the CD16 Fc receptor, a process referred to as antibody-dependent cell-mediated cytotoxicity (ADCC).91 NK cell development, proliferation, and cytotoxicity are enhanced by cytokines produced by T cells (IL-2, IFN-y, APCs (IL-12), and nonhematopoietic cells, such as bone marrow mesenchymal cells (IL-15 and stem cell factor). NK cells are also important producers of IFN-y and TNF-a in the early phase of the immune response to viruses, and these cytokines may promote the development of CD4 T cells into Th-1 effector cells.51IFN-y produced by NK cells may also enhance the ability of B cells to respond to multivalent antigens, such as complex polysaccharides.160Cytokines produced by other cell types, such as IL-2 from T cells and IL-12 from dendritic cells and Mq, may be important triggers of cytokine production by NK cells.

+,

NK Cells and Their Function in the Fetus and Neonate

Cells with an NK cell-like surface phenotype predominate in the fetal liver mononuclear cell compartment, in which they are detected as early as 6 weeks of gestation and become increasingly abundant during the second trimester.lZ6Unlike circulating or adult neonatal NK cells, a

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substantial proportion of these fetal liver NK cells lack CD16 surface expression.126NK cells are present in greater numbers in the second trimester fetal circulation than s~bsequently,'~~ and their number in the neonatal circulation is typically about twofold greater than in adult^."^,'^^ In contrast to adult NK cells, virtually all neonatal and fetal NK cells lack expression of CD57, and the fraction that express CD56 is reduced by about 500/o. 55, 126 NK cells from the fetus or premature infant have reduced cytotoxic function compared with those of the term neonate.'07,126 Decreased cytotoxic activity by neonatal NK cells compared with adult cells is also consistently observed with HSV-infected target cells and with some tumor cell lines, but not In contrast, both neonatal and adult NK cells have equivalent cytotoxic activity against HIV-1 infected cells.82,lo7 The mechanisms underlying these pathogen-related differences in natural cytotoxicity during human development remains unclear. Reduced cytolytic activity parallels the reduced numbers of CD56+ NK cells in the neonate, consistent with their usually poor cytolytic a~tivity.5~ When only CD56+ neonatal NK cells are studied, their cytolytic activity is 149 similar to that of adult NK cells.'26* Like adult NK cells, IL-2, IL-12, and IFN-a, -p and -y can rapidly (within hours) augment the cytolytic activity of neonatal NK Consistent with these findings, neonatal NK cells have surface levels of receptors for IL-2 and IFN-y that are similar to or greater than those of adult NK cells.69Paralleling the reduction in natural cytotoxic activity of circulating neonatal mononuclear cells, the activity of these cells in assays measuring ADCC is also reduced. ADCC is reduced approximately 50% compared with adult mononuclear cells, including with HIV-infected targets.lo7Circulating neonatal NK cells acquire substantially greater natural cytotoxic activity when they are incubated overnight to several days with IL-2 or IL-12 to generate lymphokine-activated killer (LAK) cells.55,72, 85, Io7 This enhanced cytotoxic activity, which suggests that neonatal NK cells have a normal capacity to be primed by exogenous cytokines, is accompanied by an increased expression of CD56 by neonatal NK cells.'04Whether this expression reflects the expansion of a preexisting CD56+ NK population or the differentiation of CD56 - cells into CD56 + NK cells remains to be determined. Neonatal NK cells produce IFN-y as effectively as adult NK cells in response to exogenous IL-2 and HSV. Conflicting results have been obtained as to whether IL-12-induced production of IFN-y by neonatal mononuclear cells (most likely mediated by NK cells) is reduced com96; additional studies using purified NK cell pared with adult cells92* populations may be informative. Whether the production of other cytokines by neonatal NK cells, particularly with stimuli other than HSV, is reduced compared with adult NK cells remains to be determined. Congenital viral or Toxoplasma infection during the second trimester may result in a substantial increase in the number of circulating NK cells,'74 suggesting that fetal NK cells can respond to infections to some degree. In such cases, persistent increases in NK cells can persist until

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birth and may be accompanied by decreased NK cell expression of CD45RA and increased expression of CD45R0.l1O This CD45RALoW CD45RO'"ph surface phenotype suggests that these cells have been activated in vivo because similar alterations occur when NK cells are incubated in vitro with either IL-2 or tumor cell targets.16Whether congenital infection results in enhanced natural cytotoxicity by fetal or neonatal NK cells remains to be determined.

CONCLUSION

Limitations in the available repertoire of a/p T-cell receptors appear unlikely to play a major role in limiting responses to infection by the fetus by mid-gestation. However, there is substantial evidence that multiple aspects of T-cell function in the fetus and neonate are impaired compared with that in adults, including cell-mediated cytotoxicity, antigen-specific cytokine production, such as for IFN-7, and help for B-cell antibody responses. Selective decreases in the production of cytokines by neonatal T cells, such as the CD40-ligand, compared with adult antigenically naive T cells, may contribute to these deficits, particularly for reduced antibody responses. It is also possible that relatively subtle but important decreases in APC function, for example by dendritic cells, may also contribute to the poor outcome of some T-cell and B-cell responses. Most recent studies suggest that the intrinsic capacity for fetal and neonatal B cells to be activated and undergo isotype switching is similar to that of antigenically naive B cells in the adult. However, the adequacy of these responses in vitro does not exclude the possibility that B cells of the fetus may be more prone to tolerance induction in vivo than antigenically naive B cells that subsequently appear after birth. Although NK cells appear early during gestation and are present in normal numbers by mid to late gestation, the phenotype of approximately 50% portion of these cells is immature (CD56- ). These immature cells have decreased cytotoxic activity compared with cells from adults, which are uniformly CD56+. This immaturity is associated with decreased functional activity, including diminished natural cytotoxic activity against cells infected with herpes group viruses.

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