免疫学（英文版）：Chapter 09

identify and isolate its antigen-binding receptor. The obvi- ous parallels between the recognition functions of T cells and B cells stimulated a great deal of experimental effort to take advantage of the anticipated structural similarities be- tween immunoglobulins and T-cell receptors. Reports published in the 1970s claimed discovery of immunoglob- ulin isotypes associated exclusively with T cells (IgT) and of antisera that recognize variable-region markers (idio- types) common to antibodies and T-cell receptors with similar specificity. These experiments could not be repro- duced and were proven to be incorrect when it was demon- strated that the T-cell receptor and immunoglobulins do not have common recognition elements and are encoded by entirely separate gene families. As the following sections will show, a sequence of well-designed experiments using cutting-edge technology was required to correctly answer questions about the structure of the T-cell receptor, the genes that encode it, and the manner in which it recognizes antigen. chapter 9 a73 Early Studies of the T-Cell Receptor a73 H9251H9252 and H9253H9254 T-Cell Receptors: Structure and Roles a73 Organization and Rearrangement of TCR Genes a73 T-Cell Receptor Complex: TCR-CD3 a73 T-Cell Accessory Membrane Molecules a73 Three-Dimensional Structures of TCR-Peptide- MHC Complexes a73 Alloreactivity of T Cells T-Cell Receptor T ?? ???????-???????? ?????? ?? ?-???? ????????? clearly implies that T cells possess an antigen- specific and clonally restricted receptor. However, the identity of this receptor remained unknown long after the B-cell receptor (immunoglobulin molecule) had been identified. Relevant experimental results were contradictory and difficult to conceptualize within a single model because the T-cell receptor (TCR) differs from the B-cell antigen- binding receptor in important ways. First, the T-cell receptor is membrane bound and does not appear in a soluble form as the B-cell receptor does; therefore, assessment of its struc- ture by classic biochemical methods was complicated, and complex cellular assays were necessary to determine its speci- ficity. Second, most T-cell receptors are specific not for anti- gen alone but for antigen combined with a molecule encoded by the major histocompatibility complex (MHC). This prop- erty precludes purification of the T-cell receptor by simple antigen-binding techniques and adds complexity to any ex- perimental system designed to investigate the receptor. A combination of immunologic, biochemical, and molecular-biological manipulations has overcome these problems. The molecule responsible for T-cell specificity was found to be a heterodimer composed of either H9251 and H9252 or H9253 and H9254 chains. Cells that express TCRs have approxi- mately 10 5 TCR molecules on their surface. The genomic organization of the T-cell receptor gene families and the means by which the diversity of the component chains is generated were found to resemble those of the B-cell re- ceptor chains. Further, the T-cell receptor is associated on the membrane with a signal-transducing complex, CD3, whose function is similar to that of the Ig-H9251/Ig-H9252 complex of the B-cell receptor. Important new insights concerning T-cell receptors have been gained by recent structure determinations using x-ray crystallography, including new awareness of differences in how TCRs bind to class I or class II MHC molecules. This chapter will explore the nature of the T-cell receptor mole- cules that specifically recognize MHC-antigen complexes, as well as some that recognize native antigens. Early Studies of the T-Cell Receptor By the early 1980s, investigators had learned much about T-cell function but were thwarted in their attempts to Interaction of H9251H9252 TCR with Class II MHC–Peptide ART TO COME 8536d_ch09_200-220 8/2/02 1:00 PM Page 200 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: T-Cell Receptor CHAPTER 9 201 Classic Experiments Demonstrated the Self-MHC Restriction of the T-Cell Receptor By the early 1970s, immunologists had learned to generate cytotoxic T lymphocytes (CTLs) specific for virus-infected target cells. For example, when mice were infected with lym- phocytic choriomeningitis (LCM) virus, they would produce CTLs that could lyse LCM-infected target cells in vitro. Yet these same CTLs failed to bind free LCM virus or viral anti- gens. Why didn’t the CTLs bind the virus or viral antigens di- rectly as immunoglobulins did? The answer began to emerge in the classic experiments of R. M. Zinkernagel and P. C. Doherty in 1974 (see Figure 8-2). These studies demon- strated that antigen recognition by T cells is specific not for viral antigen alone but for antigen associated with an MHC molecule (Figure 9-1). T cells were shown to recognize anti- gen only when presented on the membrane of a cell by a self- MHC molecule. This attribute, called self-MHC restriction, distinguishes recognition of antigen by T cells and B cells. In 1996, Doherty and Zinkernagel were awarded the Nobel Prize for this work. Two models were proposed to explain the MHC restric- tion of the T-cell receptor. The dual-receptor model envi- sioned a T cell with two separate receptors, one for antigen and one for class I or class II MHC molecules. The altered-self model proposed that a single receptor recognizes an alter- ation in self-MHC molecules induced by their association with foreign antigens. The debate between proponents of these two models was waged for a number of years, until an elegant experiment by J. Kappler and P. Marrack demon- strated that specificity for both MHC and antigen resides in a single receptor. An overwhelming amount of structural and functional data has since been added in support of the altered-self model. T-Cell Receptors Were Isolated by Using Clonotypic Antibodies Identification and isolation of the T-cell receptor was accom- plished by producing large numbers of monoclonal antibod- ies to various T-cell clones and then screening the antibodies to find one that was clone specific, or clonotypic. This ap- proach assumes that, since the T-cell receptor is specific for both an antigen and an MHC molecule, there should be sig- nificant structural differences in the receptor from clone to clone; each T-cell clone should have an antigenic marker similar to the idiotype markers that characterize monoclonal antibodies. Using this approach, researchers in the early 1980s isolated the receptor and found that it was a het- erodimer consisting of H9251 and H9252 chains. When antisera were prepared using H9251H9252 heterodimers iso- lated from membranes of various T-cell clones, some antis- era bound to H9251H9252 heterodimers from all the clones, whereas other antisera were clone specific. This finding suggested that the amino acid sequences of the TCR H9251 and H9252 chains, like those of the immunoglobulin heavy and light chains, have constant and variable regions. Later, a second type of TCR heterodimer consisting of H9254 and H9253 chains was identified. In human and mouse, the majority of T cells express the H9251H9252het- erodimer; the remaining T cells express the H9253H9254 heterodimer. As described below, the exact proportion of T cells expressing H9251H9252 or H9253H9254 TCRs differs by organ and species, but H9251H9252 T cells normally predominate. The TCR H9252-Chain Gene Was Cloned by Use of Subtractive Hybridization In order to identify and isolate the TCR genes, S. M. Hedrick and M. M. Davis sought to isolate mRNA that encodes the H9251 and H9252 chains from a T H -cell clone. This was no easy task be- cause the receptor mRNA represents only a minor fraction of the total cell mRNA. By contrast, in the plasma cell, im- munoglobulin is a major secreted cell product, and mRNAs encoding the heavy and light chains are abundant and easy to purify. The successful scheme of Hedrick and Davis assumed that the TCR mRNA—like the mRNAs that encode other integral membrane proteins—would be associated with membrane- bound polyribosomes rather than with free cytoplasmic ri- bosomes. They therefore isolated the membrane-bound polyribosomal mRNA from a T H -cell clone and used reverse transcriptase to synthesize 32 P-labeled cDNA probes (Figure 9-2). Because only 3% of lymphocyte mRNA is in the membrane-bound polyribosomal fraction, this step elimi- nated 97% of the cell mRNA. Hedrick and Davis next used a technique called DNA sub- tractive hybridization to remove from their preparation the [ 32 P]cDNA that was not unique to T cells. Their rationale for this step was based on earlier measurements by Davis show- ing that 98% of the genes expressed in lymphocytes are com- mon to B cells and T cells. The 2% of the expressed genes that H-2 k CTL TCR H-2 k target cell Killing Viral peptide A Self MHC H-2 k CTL TCR H-2 d target cell No killing Viral peptide A Nonself MHC H-2 k CTL TCR H-2 k target cell No killing Viral peptide B Self MHC (a) (b) (c) FIGURE 9-1 Self-MHC restriction of the T-cell receptor (TCR). A particular TCR is specific for both an antigenic peptide and a self- MHC molecule. In this example, the H-2 k CTL is specific for viral pep- tide A presented on an H-2 k target cell (a). Antigen recognition does not occur when peptide B is displayed on an H-2 k target cell (b) nor when peptide A is displayed on an H-2 d target cell (c). 8536d_ch09_200-220 8/2/02 9:49 AM Page 201 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: represented the T-cell receptor, all were used as probes to look for genes that rearranged in mature T cells. This ap- proach was based on the assumption that, since the H9251H9252 T-cell receptor appeared to have constant and variable regions, its genes should undergo DNA rearrangements like those ob- served in the Ig genes of B cells. The two investigators tested DNA from T cells, B cells, liver cells, and macrophages by Southern-blot analysis using the 10 [ 32 P]cDNA probes to identify unique T-cell genomic DNA sequences. One clone showed bands indicating DNA rearrangement in T cells but not in the other cell types. This cDNA probe identified six different patterns for the DNA from six different mature T- cell lines (see Figure 9-2 inset, upper panel). These different patterns presumably represented rearranged TCR genes. Such results would be expected if rearranged TCR genes oc- cur only in mature T cells. The observation that each of the six T-cell lines showed different Southern-blot patterns was consistent with the predicted differences in TCR specificity in each T-cell line. The cDNA clone 1 identified by the Southern-blot analy- ses shown in Figure 9-2 has all the hallmarks of a putative TCR gene: it represents a gene sequence that rearranges, is expressed as a membrane-bound protein, and is expressed only in T cells. This cDNA clone was found to encode the H9252 chain of the T-cell receptor. Later, cDNA clones were identi- fied encoding the H9251 chain, the H9253 chain, and finally the H9254 chain. These findings opened the way to understanding the T-cell receptor and made possible subsequent structural and func- tional studies. H9251H9252 and H9253H9254 T-Cell Receptors: Structure and Roles The domain structures of H9251H9252 and H9253H9254 TCR heterodimers are strikingly similar to that of the immunoglobulins; is unique to T cells should include the genes encoding the T- cell receptor. Therefore, by hybridizing B-cell mRNA with their T H -cell [ 32 P]cDNA, they were able to remove, or sub- tract, all the cDNA that was common to B cells and T cells. The unhybridized [ 32 P]cDNA remaining after this step pre- sumably represented the expressed polyribosomal mRNA that was unique to the T H -cell clone, including the mRNA encoding its T-cell receptor. Cloning of the unhybridized [ 32 P]cDNA generated a li- brary from which 10 different cDNA clones were identified. To determine which of these T-cell–specific cDNA clones 202 PART II Generation of B-Cell and T-Cell Responses Hybridize mRNA 97% in free cytoplasmic polyribosomes 3% in membrane-bound polyribosomes 32 P Reverse transcriptase [ 32 P] cDNA mRNA cDNAs specific to T cells Hybrids with cDNAs common to T cells and B cells Separate on hydroxyapatite column 10 different cDNA clones Liver cells B - cell lymphoma abcdef Probed with cDNA clone 1 Probed with cDNA clone 2 T-cell clones T H -cell clone B cell Use as probes in Southern blots of genomic DNA FIGURE 9-2 Production and identification of a cDNA clone en- coding the T-cell receptor. The flow chart outlines the procedure used by S. Hedrick and M. Davis to obtain [ 32 P]cDNA clones correspond- ing to T-cell–specific mRNAs. The technique of DNA subtractive hy- bridization enabled them to isolate [ 32 P]cDNA unique to the T cell. The labeled T H -cell cDNA clones were used as probes (inset) in Southern-blot analyses of genomic DNA from liver cells, B-lym- phoma cells, and six different T H -cell clones (a–f). Probing with cDNA clone 1 produced a distinct blot pattern for each T-cell clone, whereas probing with cDNA clone 2 did not. Assuming that liver cells and B cells contained unrearranged germ-line TCR DNA, and that each of the T-cell clones contained different rearranged TCR genes, the results using cDNA clone 1 as the probe identified clone 1 as the T-cell–receptor gene. The cDNA of clone 2 identified the gene for another T-cell membrane molecule encoded by DNA that does not undergo rearrangement. [Based on S. Hedrick et al., 1984, Nature 308:149.] 8536d_ch09_200-220 8/2/02 9:49 AM Page 202 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: thus, they are classified as members of the immunoglobulin superfamily (see Figure 4-19). Each chain in a TCR has two domains containing an intrachain disulfide bond that spans 60–75 amino acids. The amino-terminal domain in both chains exhibits marked sequence variation, but the sequences of the remainder of each chain are conserved. Thus the TCR domains–one variable (V) and one constant (C)–are struc- turally homologous to the V and C domains of immuno- globulins, and the TCR molecule resembles an Fab fragment (Figure 9-3). The TCR variable domains have three hyper- variable regions, which appear to be equivalent to the complementarity determining regions (CDRs) in immuno- globulin light and heavy chains. There is an additional area of hypervariability (HV4) in the H9252 chain that does not normally contact antigen and therefore is not considered a CDR. In addition to the constant domain, each TCR chain con- tains a short connecting sequence, in which a cysteine residue forms a disulfide link with the other chain of the het- erodimer. Following the connecting region is a transmem- brane region of 21 or 22 amino acids, which anchors each chain in the plasma membrane. The transmembrane do- mains of both chains are unusual in that they contain posi- tively charged amino acid residues. These residues enable the chains of the TCR heterodimer to interact with chains of the signal-transducing CD3 complex. Finally, each TCR chain T-Cell Receptor CHAPTER 9 203 contains a short cytoplasmic tail of 5–12 amino acids at the carboxyl-terminal end. H9251H9252 and H9253H9254 T-cell receptors were initially difficult to inves- tigate because, like all transmembrane proteins, they are in- soluble. This problem was circumvented by expressing modified forms of the protein in vitro that had been engi- neered to contain premature in-frame stop codons that pre- clude translation of the membrane-binding sequence that makes the molecule insoluble. The majority of T cells in the human and the mouse ex- press T-cell receptors encoded by the H9251H9252 genes. These recep- tors interact with peptide antigens processed and presented on the surface of antigen-presenting cells. Early indications that certain T cells reacted with nonpeptide antigens were puzzling until some light was shed on the problem when products of the CD1 family of genes were found to present carbohydrates and lipids. More recently, it has been found that certain H9253H9254 cells react with antigen that is neither processed nor presented in the context of a MHC molecules. Differences in the antigen-binding regions of H9251H9252 and H9253H9254 were expected because of the different antigens they recog- nize, but no extreme dissimilarities were expected. However, the recently completed three-dimensional structure for a H9253H9254 receptor that reacts with a phosphoantigen, reported by Allison, Garboczi, and their coworkers, reveals significant NH 2 NH 2 V α V L V L C L C L V H V H C μ C μ β-chainα-chain S S S S S S S S S S NH 2 NH 2 + + + αβ T-cell receptor B-cell mIgM L chains H chainH chain Connecting sequence Transmembrane region (T m ) Cytoplasmic tail (CT) C μ V β C μ C α C μ C β C μ S S S S S S S S S S C μ C μ S S S S S S S S S S S S S S S S S S S S S S S S COOH (248) COOH (282) FIGURE 9-3 Schematic diagram illustrating the structural similar- ity between the H9251H9252 T-cell receptor and membrane-bound IgM on B cells. The TCR H9251and H9252chain each contains two domains with the im- munoglobulin-fold structure. The amino-terminal domains (V H9251 and V H9252 ) exhibit sequence variation and contain three hypervariable re- gions equivalent to the CDRs in antibodies. The sequence of the con- stant domains (C H9251 and C H9252 ) does not vary. The two TCR chains are connected by a disulfide bond between their constant sequences; the IgM H chains are connected to one another by a disulfide bond in the hinge region of the H chain, and the L chains are connected to the H chains by disulfide links between the C termini of the L chains and the C H9262 region. TCR molecules interact with CD3 via positively charged amino acid residues (indicated by H11001) in their transmem- brane regions. Numbers indicate the length of the chains in the TCR molecule. Unlike the antibody molecule, which is bivalent, the TCR is monovalent. 8536d_ch09_200-220 8/2/02 9:49 AM Page 203 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: differences in the overall structures of the two receptor types, pointing to possible functional variation. The receptor they studied was composed of the H92539 and H92542 chains, which are those most frequently expressed in human peripheral blood. A deep cleft on the surface of the molecule accommodates the microbial phospholipid for which the H9253H9254 receptor is spe- cific. This antigen is recognized without MHC presentation. The most striking feature of the structure is how it differs from the H9251H9252 receptor in the orientation of its V and C re- gions. The so-called elbow angle between the long axes of the V and C regions of H9253H9254 TCR is 111°; in the H9251H9252 TCR, the elbow angle is 149°, giving the molecules distinct shapes (Figure 9-4). The full significance of this difference is not known, but it could contribute to differences in signaling mecha- nisms and in how the molecules interact with coreceptor molecules. The number of H9253H9254 cells in circulation is small compared with cells that have H9251H9252 receptors, and the V gene segments of H9253H9254 receptors exhibit limited diversity. As seen from the data in Table 9-1, the majority of H9253H9254 cells are negative for both CD4 and CD8, and most express a single H9253H9254-chain subtype. In humans the predominant receptor expressed on circulat- ing H9253H9254 cells recognizes a microbial phospholipid antigen, 3- formyl-1-butyl pyrophosphate, found on M. tuberculosis and other bacteria and parasites. This specificity for frequently encountered pathogens led to speculation that H9253H9254 cells may function as an arm of the innate immune response, allowing rapid reactivity to certain antigens without the need for a processing step. Interestingly, the specificity of circulating H9253H9254 cells in the mouse and of other species studied does not par- allel that of humans, suggesting that the H9253H9254 response may be directed against pathogens commonly encountered by a given species. Furthermore, data indicating that H9253H9254 cells can secrete a spectrum of cytokines suggest that they may play a regulatory role in recruiting H9251H9252 T cells to the site of invasion by pathogens. The recruited H9251H9252 T cells would presumably display a broad spectrum of receptors; those with the highest affinity would be selectively activated and amplified to deal with the pathogen. Organization and Rearrangement of TCR Genes The genes that encode the H9251H9252 and H9253H9254 T-cell receptors are ex- pressed only in cells of the T-cell lineage. The four TCR loci (H9251,H9252, H9253, and H9254) are organized in the germ line in a manner that is remarkably similar to the multigene organization of the immunoglobulin (Ig) genes (Figure 9-5). As in the case of Ig genes, functional TCR genes are produced by re- arrangements of V and J segments in the H9251-chain and H9253- chain families and V, D, and J segments in the H9252-chain and H9254-chain families. In the mouse, the H9251-, H9252-, and H9253-chain gene segments are located on chromosomes 14, 6, and 13, respec- tively. The H9254-gene segments are located on chromosome 14 between the V H9251 and J H9251 segments. The location of the H9254-chain gene family is significant: a productive rearrangement of the H9251-chain gene segments deletes C H9254 , so that, in a given T cell, the H9251H9252 TCR receptor cannot be coexpressed with the H9253H9254 receptor. Mouse germ-line DNA contains about 100 V H9251 and 50 J H9251 gene segments and a single C H9251 segment. The H9254-chain gene family contains about 10 V gene segments, which are largely distinct from the V H9251 gene segments, although some sharing 204 PART II Generation of B-Cell and T-Cell Responses V domains C domains H9253H9254 TCR 111H11034 H9251H9252 TCR 147H11034 FIGURE 9-4 Comparison of the H9253H9254 TCR and H9251H9252 TCR. The dif- ference in the elbow angle is highlighted with black lines. [From T. Allison et al., 2001, Nature 411: 820.] TABLE 9-1 Comparison of H9251H9252 and H9253H9254 T cells Feature H9251H9252 T cells H9253H9254 T cells Proportion of CD3 H11001 90–99% 1–10% cells TCR V gene germ- Large Small line repertoire CD4/CD8 phenotype CD4 H11001 ~60% H110211% CD8 H11001 ~30%~30% CD4 H11001 CD8 H11001 H110211% H110211% CD4 – CD8 – H110211%~60% MHC restriction CD4 H11001 : MHC No MHC class II restriction CD8 H11001 : MHC class I Ligands Peptide H11001 MHC Phospholipid antigen SOURCE: D. Kabelitz et al., 1999, Springer Seminars in Immunopathology 21:55, p. 36. 8536d_ch09_200-220 8/2/02 1:00 PM Page 204 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: of V segments has been observed in rearranged H9251- and H9254-chain genes. Two D H9254 and two J H9254 gene segments and one C H9254 segment have also been identified. The H9252-chain gene family has 20–30 V gene segments and two almost identical repeats of D, J, and C segments, each repeat consisting of one D H9252 , six J H9252 , and one C H9252 . The H9253-chain gene family consists of seven V H9253 segments and three different functional J H9253 -C H9253 repeats. The organization of the TCR multigene families in humans is generally similar to that in mice, although the number of seg- ments differs (Table 9-2). TCR Variable-Region Genes Rearrange in a Manner Similar to Antibody Genes The H9251 chain, like the immunoglobulin L chain, is encoded by V, J, and C gene segments. The H9252 chain, like the im- munoglobulin H chain, is encoded by V, D, J, and C gene seg- ments. Rearrangement of the TCR H9251- and H9252-chain gene segments results in VJ joining for the H9251 chain and VDJ join- ing for the H9252 chain (Figure 9-6). After transcription of the rearranged TCR genes, RNA processing, and translation, the H9251 and H9252 chains are expressed as a disulfide-linked heterodimer on the membrane of the T cell. Unlike immunoglobulins, which can be membrane bound or secreted, the H9251H9252 heterodimer is expressed only in a membrane-bound form; thus, no differential RNA process- ing is required to produce membrane and secreted forms. Each TCR constant region includes a connecting sequence, a transmembrane sequence, and a cytoplasmic sequence. The germ-line DNA encoding the TCR H9251 and H9252 chain constant regions is much simpler than the immunoglobulin heavy-chain germ-line DNA, which has multiple C gene seg- ments encoding distinct isotypes with different effector func- tions. TCR H9251-chain DNA has only a single C gene segment; the H9252-chain DNA has two C gene segments, but their protein products differ by only a few amino acids and have no known functional differences. MECHANISM OF TCR DNA REARRANGEMENTS The mechanisms by which TCR germ-line DNA is re- arranged to form functional receptor genes appear to be T-Cell Receptor CHAPTER 9 205 FIGURE 9-5 Germ-line organization of the mouse TCR H9251-, H9252-, H9253-, and H9254-chain gene segments. Each C gene segment is composed of a series of exons and introns, which are not shown. The organization of TCR gene segments in humans is similar, although the number of the various gene segments differs in some cases (see Table 9-2). [Adapted from D. Raulet, 1989, Annu. Rev. Immunol. 7:175, and M. Davis, 1990, Annu. Rev. Biochem. 59:475.] 3′5′ V α 1L Mouse TCR α-chain and δ-chain DNA (chromosome 14) L D δ 1V α 2 V α nLL L D δ 2J δ 1J δ 2C δ L J α 1V δ 5J α 2J α 3 J α n C α V δ nV δ 1 3′5′ V β 1L Mouse TCR β-chain DNA (chromosome 6) L V β 2L J β 1.1?J β 1.7 C β 1V β n D β 1D β 2C β 2LV β 14 3′5′ V γ 5L Mouse TCR γ-chain DNA (chromosome 13) L V γ 2L C γ 1V γ 4 C γ 2L V γ 3 L L V γ 1.1 J γ 4 V γ 1.2 C γ 4LJ γ 2C γ 3J γ 3J γ 1 V γ 1.3 ψψ ψ ψ J β 2.1?J β 2.7 ψ (V α n = ~100 ; V δ n = ~10) (V β n = 20 ? 30) (J α n = ~50) = Enhancer = pseudogene TABLE 9-2 TCR Multigene families in humans NO. OF GENE SEGMENTS Chromosome Gene location V D J C H9251 Chain 14 50 70 1 H9254 Chain * 14 33 31 H9252 Chain ? 7572132 H9253 Chain ? 714 5 * The H9254-chain gene segments are located between the V H9251 and J H9251 segments. ? There are two repeats, each containing 1 D H9252 , 6 or 7 J H9252 , and 1 C H9252 . ? There are two repeats, each containing 2 or 3 J H9253 and 1 C H9253 . SOURCE: Data from P. A. H. Moss et al., 1992, Annu. Rev. Immunol. 10:71. 8536d_ch09_200-220 8/2/02 9:49 AM Page 205 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: similar to the mechanisms of Ig-gene rearrangements. For example, conserved heptamer and nonamer recombination signal sequences (RSSs), containing either 12-bp (one-turn) or 23-bp (two-turn) spacer sequences, have been identified flanking each V, D, and J gene segment in TCR germ-line DNA (see Figure 5-6). All of the TCR-gene rearrangements follow the one-turn/two-turn joining rule observed for the Ig genes, so recombination can occur only between the two dif- ferent types of RSSs. Like the pre-B cell, the pre-T cell expresses the recombi- nation-activating genes (RAG-1 and RAG-2). The RAG-1/2 recombinase enzyme recognizes the heptamer and non- amer recognition signals and catalyzes V-J and V-D-J join- ing during TCR-gene rearrangement by the same deletional or inversional mechanisms that occur in the Ig genes (see Figure 5-7). As described in Chapter 5 for the immunoglobulin genes, RAG-1/2 introduces a nick on one DNA strand between the coding and signal sequences. The recombinase then catalyzes a transesterification reaction that results in the formation of a hairpin at the coding sequence and a flush 5H11032 phosphorylated double-strand break at the signal sequence. Circular excision products thought to be generated by looping-out and deletion dur- ing TCR-gene rearrangement have been identified in thy- mocytes (see Figure 5-8). Studies with SCID mice, which lack functional T and B cells, provide evidence for the similarity in the mechanisms of Ig-gene and TCR-gene rearrangements. As explained in Chapter 19, SCID mice have a defect in a gene required for the repair of double-stranded DNA breaks. As a result of this defect, D and J gene segments are not joined during re- arrangement of either Ig or TCR DNA (see Figure 5-10). This 206 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS FIGURE 9-6 Example of gene rearrangements that yield a func- tional gene encoding the H9251H9252 T-cell receptor. The H9251-chain DNA, analogous to immunoglobulin light-chain DNA, undergoes a variable-region V H9251 -J H9251 joining. The H9252-chain DNA, analogous to im- munoglobulin heavy-chain DNA, undergoes two variable-region joinings: first D H9252 to J H9252 and then V H9252 to D H9252 J H9252 . Transcription of the re- arranged genes yields primary transcripts, which are processed to give mRNAs encoding the H9251 and H9252 chains of the membrane- bound TCR. The leader sequence is cleaved from the nascent polypeptide chain and is not present in the finished protein. As no secreted TCR is produced, differential processing of the pri- mary transcripts does not occur. Although the H9252-chain DNA con- tains two C genes, the gene products of these two C genes exhibit no known functional differences. The C genes are composed of several exons and introns, which are not individually shown here (see Figure 9-7). Protein product αβ heterodimer 3′ 5′ V α 1 C δ V α n D δ 1D δ 2 J δ 1J δ 2 C α J α 1J α 2 J α n Germ-line α-chain DNA 3′5′ V α 1V α 2J α nV α T cell J α C α Rearranged α-chain DNA L L L V α J α C α S S V β D β J β C β L 3′5′ V β 1V β Rearranged β-chain DNA L L L V β n V δ nLV δ 1LV δ 5L D β J β D β 2C β C β 2 V β 14L Germ-line β-chain DNA 3′5′ V β L J β J β D β 2 C β 2V β 14LL J β D β C β 1 8536d_ch09_200-220 8/2/02 9:49 AM Page 206 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: finding suggests that the same double-stranded break-repair enzymes are involved in V-D-J rearrangements in B cells and in T cells. Although B cells and T cells use very similar mechanisms for variable-region gene rearrangements, the Ig genes are not normally rearranged in T cells and the TCR genes are not re- arranged in B cells. Presumably, the recombinase enzyme sys- tem is regulated in each cell lineage, so that only rearrangement of the correct receptor DNA occurs. Re- arrangement of the gene segments in both T and B cell cre- ates a DNA sequence unique to that cell and its progeny. The large number of possible configurations of the rearranged genes makes this new sequence a marker that is specific for the cell clone. These unique DNA sequences have been used to aid in diagnoses and in treatment of lymphoid leukemias and lymphomas, cancers that involve clonal proliferation of T or B cells (see Clinical Focus on page 208). ALLELIC EXCLUSION OF TCR GENES As mentioned above, the H9254 genes are located within the H9251- gene complex and are deleted by H9251-chain rearrangements. This event provides an irrevocable mode of exclusion for the H9254 genes located on the same chromosome as the rearranging H9251 genes. Allelic exclusion of genes for the TCR H9251 and H9252 chains occurs as well, but exceptions have been observed. The organization of the H9252-chain gene segments into two clusters means that, if a nonproductive rearrangement oc- curs, the thymocyte can attempt a second rearrangement. This increases the likelihood of a productive rearrangement for the H9252 chain. Once a productive rearrangement occurs for one H9252-chain allele, the rearrangement of the other H9252 allele is inhibited. Exceptions to allelic exclusion are most often seen for the TCR H9251-chain genes. For example, analyses of T-cell clones that express a functional H9251H9252 T-cell receptor revealed a num- ber of clones with productive rearrangements of both H9251- chain alleles. Furthermore, when an immature T-cell lymphoma that expressed a particular H9251H9252 T-cell receptor was subcloned, several subclones were obtained that expressed the same H9252-chain allele but an H9251-chain allele different from the one expressed by the original parent clone. Studies with transgenic mice also indicate that allelic exclusion is less stringent for TCR H9251-chain genes than for H9252-chain genes. Mice that carry a productively rearranged H9251H9252-TCR transgene do not rearrange and express the endogenous H9252-chain genes. However, the endogenous H9251-chain genes sometimes are ex- pressed at various levels in place of the already rearranged H9251- chain transgene. Since allelic exclusion is not complete for the TCR H9251 chain, there are rare occasions when more than one H9251 chain is expressed on the membrane of a given T cell. The obvious question is how do the rare T cells that express two H9251H9252 T-cell receptors maintain a single antigen-binding specificity? One proposal suggests that when a T cell expresses two different H9251H9252 T-cell receptors, only one is likely to be self-MHC re- stricted and therefore functional. Rearranged TCR Genes Are Assembled from V, J, and D Gene Segments The general structure of rearranged TCR genes is shown in Figure 9-7. The variable regions of T-cell receptors are, of course, encoded by rearranged VDJ and VJ sequences. In TCR genes, combinatorial joining of V gene segments ap- pears to generate CDR1 and CDR2, whereas junctional flexi- bility and N-region nucleotide addition generate CDR3. Rearranged TCR genes also contain a short leader (L) exon upstream of the joined VJ or VDJ sequences. The amino acids encoded by the leader exon are cleaved as the nascent polypeptide enters the endoplasmic reticulum. The constant region of each TCR chain is encoded by a C gene segment that has multiple exons (see Figure 9-7) corre- sponding to the structural domains in the protein (see Figure 9-3). The first exon in the C gene segment encodes most of the C domain of the corresponding chain. Next is a short exon that encodes the connecting sequence, followed by ex- ons that encode the transmembrane region and the cytoplas- mic tail. TCR Diversity Is Generated Like Antibody Diversity but Without Somatic Mutation Although TCR germ-line DNA contains far fewer V gene seg- ments than Ig germ-line DNA, several mechanisms that op- erate during TCR gene rearrangements contribute to a high degree of diversity among T-cell receptors. Table 9-3 (page 210) and Figure 9-8 (page 211) compare the generation of diversity among antibody molecules and TCR molecules. T-Cell Receptor CHAPTER 9 207 Rearranged α-chain gene Rearranged β-chain gene V α C α C β C α H Tm CT V Encoded domains L J CDR1 CDR2 CDR3 V β C β H Tm CT V L J CDR1 CDR2 CDR3 Leader Variable domain (V α or V β ) Constant domain (C α or C β ) Connecting sequence Trans- membrane region Cyto- plasmic tail D FIGURE 9-7 Schematic diagram of rearranged H9251H9252-TCR genes showing the exons that encode the various domains of the H9251H9252 T-cell receptor and approximate position of the CDRs. Junctional diversity (vertical arrows) generates CDR3 (see Figure 9-8). The structures of the rearranged H9253- and H9254-chain genes are similar, although additional junctional diversity can occur in H9254-chain genes. 8536d_ch09_200-220 8/2/02 2:06 PM Page 207 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: 208 PART II Generation of B-Cell and T-Cell Responses the TCR genes in the T cells occurs be- fore the product molecule is expressed, T cells in early stages of development can be detected. The unique gene frag- ments that result from TCR gene re- arrangement can be detected by sim- ple molecular-biological techniques and provide a true fingerprint for a clonal cell population. DNA patterns that result from re- arrangement of the genes in the TCR H9252 region are used most frequently as mark- ers. There are approximately 50 V H9252 gene segments that can rearrange to one of two D-region gene segments and subse- quently to one of 12 J gene segments (see Figure 9-8). Because each of the 50 or so V-region genes is flanked by unique sequences, this process creates new DNA sequences that are unique to each cell that undergoes the rearrangement; these new sequences may be detected by Southern-blot techniques or by PCR (polymerase chain reaction). Since the entire sequence of the D, J, and C region of the TCR gene H9252 complex is known, the appropriate probes and restriction en- zymes are easily chosen for Southern blotting (see diagram). Detection of rearranged TCR DNA may be used as a diagnostic tool when abnormally enlarged lymph nodes per- sist; this condition could result either from inflammation due to chronic infec- tion or from proliferation of a cancerous lymphoid cell. If inflammation is the cause, the cells would come from a vari- ety of clones, and the DNA isolated from them would be a mixture of many different TCR sequences resulting from multiple rearrangements; no unique fragments would be detected. If the per- sistent enlargement of the nodes repre- sents a clonal proliferation, there would be a detectable DNA fragment, because the cancerous cells would all contain the same TCR DNA sequence produced by DNA rearrangement in the parent cell. Thus the question whether the ob- served enlargement was due to the can- cerous growth of T cells could be answered by the presence of a single new gene fragment in the DNA from the cell population. Because Ig genes re- arrange in the same fashion as the TCR genes, similar techniques use Ig probes to detect clonal B-cell populations by their unique DNA patterns. The tech- nique, therefore, has value for a wide range of lymphoid-cell cancers. Although the detection of a unique DNA fragment resulting from rearranged TCR or Ig genes indicates clonal prolifer- ation and possible malignancy of T or B cells, the absence of such a fragment does not rule out cancer of a population of lymphoid cells. The cell involved may not contain rearranged TCR or Ig genes that can be detected by the method used, either because of its developmental stage or because it is of another lineage (H9253H9254 T cells, for example). If the DNA fragment test and other di- agnostic criteria indicate that the patient has a lymphoid cell cancer, treatment by T-cell cancers, which include leukemia and lymphoma, involve the un- controlled proliferation of a clonal popu- lation of T cells. Successful treatment requires quick and certain diagnosis in order to apply the most effective treat- ment. Once treatment is initiated, reli- able tests are needed to determine whether the treatment regimen was suc- cessful. In principle, because T-cell can- cers are clonal in nature, the cell population that is cancerous could be identified and monitored by the expres- sion of its unique T-cell receptor mole- cules. However, this approach is rarely practical because detection of a specific TCR molecule requires the tedious and lengthy preparation of a specific anti- body directed against its variable region (an anti-idiotype antibody). Also, surface expression of the TCR molecule occurs somewhat late in the development of the T cell, so cancers stemming from T cells that have not progressed beyond an early stage of development will not display a TCR molecule and will not be detected by the antibody. An alternative means of identifying a clonal population of T cells is to look at their DNA rather than pro- tein products. The pattern resulting from rearrangement of the TCR genes can provide a unique marker for the cancer- ous T cell. Because rearrangement of CLINICAL FOCUS T-Cell Rearrangements as Markers for Cancerous Cells Combinatorial joining of variable-region gene segments generates a large number of random gene combinations for all the TCR chains, as it does for the Ig heavy- and light- chain genes. For example, 100 V H9251 and 50 J H9251 gene segments can generate 5 H11003 10 3 possible VJ combinations for the TCR H9251 chain. Similarly, 25 V H9252 ,2 D H9252 , and 12 J H9252 gene segments can give 6 H11003 10 2 possible combinations. Although there are fewer TCR V H9251 and V H9252 gene segments than immunoglobulin V H and V H9251 segments, this difference is offset by the greater number of J segments in TCR germ-line DNA. Assuming that the antigen-binding specificity of a given T-cell receptor depends upon the variable region in both chains, random association of 5 H11003 10 3 V H9251 combinations with 6 H11003 10 2 V H9252 combinations can generate 3 H11003 10 6 possible combinations 8536d_ch09_200-220 8/22/02 2:51 PM Page 208 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: T-Cell Receptor CHAPTER 9 209 EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI 3′5′ 12 kb 4.2 kb V β n D β 1 Rearranged β-chain DNA L LL J β D β 2C β 1 C β 2 Germ-line β-chain DNA J β V β 1 3′5′ 5 kb PCR 4.2 kb J β L J β D β 2C β 1 C β 2V β 1 Germ-line DNA Rearranged DNA Southern blot probed with C DNA 12 kb 5 kb 4.2 kb V β 2 V β 2D β Digestion of human TCR H9252-chain DNA in a germ-line (nonrearranged) configuration with EcoRI and then probing with a C-region sequence will detect the indicated C-containing frag- ments by Southern blotting. When the DNA has rearranged, a 5H11032 restriction site will be excised. Digestion with EcoRI will yield a different fragment unique to the specific V H9252 and J H9252 region gene segments incorporated into the rearranged gene, as indicated in this hypothetical example. The technique used for this analysis derives from that first used by S. M. Hedrick and his coworkers to detect unique TCR H9252 genes in a series of mouse T-cell clones (see inset to Figure 9-2). For highly sensitive detection of the rearranged TCR sequence, the polymerase chain reaction (PCR) is used. The sequence of the 5H11032 primer (red bar) is based on a unique sequence in the (V H9252 ) gene segment used by the cancerous clone (V H9252 2 in this example) and the 3H11032 primer (red bar) is a constant-region sequence. For chromosomes on which this V gene is not rearranged, the fragment will be absent because it is too large to be efficiently amplified. plify, or synthesize multiple copies of, a specific DNA sequence in a sample; primers can hybridize to the two ends of that specific sequence and thus direct a DNA polymerase to copy it; see Figure 23-13 for details.) To detect a portion of the rearranged TCR DNA, amplification using a sequence from the rearranged V region as one primer and a sequence from the H9252-chain C region as the other primer will yield a rearranged TCR DNA fragment of predicted size in sufficient quantity to be detected by electrophore- sis (see red arrow in the diagram). Re- cently, quantitative PCR methods have been used to follow patients who are in remission in order to make decisions about resuming treatment if the num- ber of cancerous cells, as estimated by these techniques, has risen above a cer- tain level. Therefore, the presence of the rearranged DNA in the clonal popula- tion of T cells gives the clinician a valu- able tool for diagnosing lymphoid-cell cancer and for monitoring the progress of treatment. radiation therapy or chemotherapy would follow. The success of this treatment can be monitored by probing DNA from the patient for the unique sequence found in the cancerous cell. If the treatment regi- men is successful, the number of cancer- ous cells will decline greatly. If the number of cancerous cells falls below 1% or 2% of the total T-cell population, analysis by Southern blot may no longer detect the unique fragment. In this case, a more sensitive technique, PCR, may be used. (With PCR it is possible to am- for the H9251H9252 T-cell receptor. Additional means to generate di- versity in the TCR V genes are described below, so 3 H11003 10 6 combinations represents a minimum estimate. As illustrated in Figure 9-8b, the location of one-turn (12-bp) and two-turn (23-bp) recombination signal se- quences (RSSs) in TCR H9252- and H9254-chain DNA differs from that in Ig heavy-chain DNA. Because of the arrangement of the RSSs in TCR germ-line DNA, alternative joining of D gene segments can occur while the one-turn/two-turn join- ing rule is observed. Thus, it is possible for a V H9252 gene seg- ment to join directly with a J H9252 or a D H9252 gene segment, generating a (VJ) H9252 or (VDJ) H9252 unit. Alternative joining of H9254-chain gene segments generates similar units; in addition, one D H9254 can join with another, 8536d_ch09_200-220 8/22/02 2:51 PM Page 209 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: random selection of nucleotides (see Figure 9-8d). Some of these combinations, however, lead to nonproductive re- arrangements by inserting in-frame stop codons that prema- turely terminate the TCR chain, or by substituting amino acids that render the product nonfunctional. Although each junctional region in a TCR gene encodes only 10–20 amino acids, enormous diversity can be generated in these regions. Estimates suggest that the combined effects of P- and N- region nucleotide addition and joining flexibility can gener- ate as many as 10 13 possible amino acid sequences in the TCR junctional regions alone. The mechanism by which diversity is generated for the TCR must allow the receptor to recognize a very large num- ber of different processed antigens while restricting its MHC-recognition repertoire to a much smaller number of self-MHC molecules. TCR DNA has far fewer V gene seg- ments than Ig DNA (see Table 9-3). It has been postulated that the smaller number of V gene segments in TCR DNA have been selected to encode a limited number of CDR1 and CDR2 regions with affinity for regions of the H9251 helices of MHC molecules. Although this is an attractive idea, it is 210 PART II Generation of B-Cell and T-Cell Responses yielding (VDDJ) H9254 and, in humans, (VDDDJ) H9254 . This mecha- nism, which cannot operate in Ig heavy-chain DNA, gener- ates considerable additional diversity in TCR genes. The joining of gene segments during TCR-gene re- arrangement exhibits junctional flexibility. As with the Ig genes, this flexibility can generate many nonproductive re- arrangements, but it also increases diversity by encoding sev- eral alternative amino acids at each junction (see Figure 9-8c). In both Ig and TCR genes, nucleotides may be added at the junctions between some gene segments during re- arrangement (see Figure 5-15). Variation in endonuclease cleavage leads to the addition of further nucleotides that are palindromic. Such P-region nucleotide addition can occur in the genes encoding all the TCR and Ig chains. Addition of N-region nucleotides, catalyzed by a terminal deoxynu- cleotidyl transferase, generates additional junctional diver- sity. Whereas the addition of N-region nucleotides in immunoglobulins occurs only in the Ig heavy-chain genes, it occurs in the genes encoding all the TCR chains. As many as six nucleotides can be added by this mechanism at each junc- tion, generating up to 5461 possible combinations, assuming TABLE 9-3 Sources of possible diversity in mouse immunoglobulin and TCR genes IMMUNOGLOBULINS H9251H9252 T-CELL RECEPTOR H9253H9254 T-CELL RECEPTOR Mechanism of diversity H Chain H9260 Chain H9251 Chain H9252 Chain H9253 Chain H9254 Chain ESTIMATED NUMBER OF SEGMENTS Multiple germ-line gene segments V 134 85 100 25 7 10 D 13 0 0 2 0 2 J 4 4 50 12 3 2 POSSIBLE NUMBER OF COMBINATIONS * Combinatorial V-J 134 H11003 13 H11003 485H11003 4 100 H11003 50 25 H11003 2 H11003 12 7 H11003 310H11003 2 H11003 2 and V-D-J joining H11005 7 H11003 10 3 H11005 3.4 H11003 10 2 H11005 5 H11003 10 3 H11005 6 H11003 10 2 H11005 21 H11005 40 Alternative joining H11002H11002H11002H11001H11002H11001 of D gene segments (some) (often) Junctional flexibility H11001H11001H11001H11001H11001H11001 N-region nucleotide addition ? H11001H11002H11001H11001H11001H11001 P-region nucleotide addition H11001H11001H11001H11001H11001H11001 Somatic mutation H11001H11001H11002H11002H11002H11002 Combinatorial association of chains H11001H11001H11001 * A plus sign (H11001) indicates mechanism makes a significant contribution to diversity but to an unknown extent. A minus sign (H11002) indicates mechanism does not operate. ? See Figure 9-8d for theoretical number of combinations generated by N-region addition. 8536d_ch09_200-220 8/22/02 2:51 PM Page 210 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: made unlikely by recent data on the structure of the TCR- peptide-MHC complex showing contact between peptide and CDR1 as well as CDR3. Therefore the TCR residues that bind to peptide versus those that bind MHC are not confined solely to the highly variable CDR3 region. In contrast to the limited diversity of CDR1 and CDR2, the CDR3 of the TCR has even greater diversity than that seen in immunoglobulins. Diversity in CDR3 is generated by junctional diversity in the joining of V, D, and J segments, joining of multiple D gene segments, and the introduction of P and N nucleotides at the V-D-J and V-J junctions (see Figure 9-7). Unlike the Ig genes, the TCR genes do not appear to un- dergo extensive somatic mutation. That is, the functional TCR genes generated by gene rearrangements during T-cell maturation in the thymus have the same sequences as those found in the mature peripheral T-cell population. The absence of somatic mutation in T cells ensures that T-cell T-Cell Receptor CHAPTER 9 211 FIGURE 9-8 Comparison of mechanisms for generating diversity in TCR genes and immunoglobulin genes. In addition to the mecha- nisms shown, P-region nucleotide addition occurs in both TCR and Ig genes, and somatic mutation occurs in Ig genes. Combinatorial association of the expressed chains generates additional diversity among both TCR and Ig molecules. T-CELL RECEPTOR (a) Combinatorial V-J and V-D-J joining IMMUNOGLOBULIN (b) Alternative joining of D gene segments (c) Junctional flexibility VJD β and δ chains VJ α and γ chains VJD H chain VJ L chain L V δ D δ D δ V δ D δ J δ V H D H J H = One-turn RSS = Two-turn RSS V δ -J δ , V δ -D δ -J δ , V δ -D δ -D δ -J δ V H -D H -J H only CACTGTG GTGGACT GATGCTCC CACAGTG One-turn RSS Two-turn RSS D H V H CACTGTG ATGGACT TGGCCG CACAGTG One-turn RSS Two-turn RSS V D J V J (d) N-region nucleotide addition V J V JD α, γ, and δ chains β and δ chains (5461) 1 = 5.5 × 10 3 (5461) 2 = 3.0 × 10 7 V JD (5461) 2 = 3.0 × 10 7 V JDD δ chain (5461) 3 = 1.6 × 10 11 = Addition of 0–6 nucleotides (5461 permutations) Heavy chain L 8536d_ch09_200-220 8/22/02 2:51 PM Page 211 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: specificity does not change after thymic selection and there- fore reduces the possibility that random mutation might generate a self-reactive T cell. Although a few experiments have provided evidence for somatic mutation of receptor genes in T cells in the germinal center, this appears to be the exception and not the rule. T-Cell Receptor Complex: TCR-CD3 As explained in Chapter 4, membrane-bound immunoglob- ulin on B cells associates with another membrane protein, the Ig-H9251/Ig-H9252 heterodimer, to form the B-cell antigen recep- tor (see Figure 4-18). Similarly, the T-cell receptor associates with CD3, forming the TCR-CD3 membrane complex. In both cases, the accessory molecule participates in signal transduction after interaction of a B or T cell with antigen; it does not influence interaction with antigen. The first evidence suggesting that the T-cell receptor is as- sociated with another membrane molecule came from ex- periments in which fluorescent antibody to the receptor was shown to cause aggregation of another membrane protein designated CD3. Later experiments by J. P. Allison and L. Lanier using cross-linking reagents demonstrated that the two chains must be within 12 ?. Subsequent experiments demonstrated not only that CD3 is closely associated with the H9251H9252heterodimer but also that its expression is required for membrane expression of H9251H9252 and H9253H9254 T-cell receptors—each heterodimer forms a complex with CD3 on the T-cell mem- brane. Loss of the genes encoding either CD3 or the TCR chains results in loss of the entire molecular complex from the membrane. CD3 is a complex of five invariant polypeptide chains that associate to form three dimers: a heterodimer of gamma and epsilon chains (H9253H9280), a heterodimer of delta and epsilon chains (H9254H9280), and a homodimer of two zeta chains (H9256H9256) or a heterodimer of zeta and eta chains (H9256H9257) (Figure 9-9). The H9256 and H9257 chains are encoded by the same gene, but differ in their carboxyl-terminal ends because of differences in RNA splicing of the primary transcript. About 90% of the CD3 complexes examined to date incorporate the (H9256H9256) homodimer; the remainder have the (H9256H9257) heterodimer. The T-cell receptor complex can thus be envi- sioned as four dimers: the H9251H9252 or H9253H9254 TCR heterodimer deter- mines the ligand-binding specificity, whereas the CD3 dimers (H9253H9280,H9254H9280, and H9256H9256 or H9256H9257) are required for membrane expression of the T-cell receptor and for signal transduction. 212 PART II Generation of B-Cell and T-Cell Responses FIGURE 9-9 Schematic diagram of the TCR-CD3 complex, which constitutes the T-cell antigen-binding receptor. The CD3 complex consists of the H9256H9256 homodimer (alternately, a H9256H9257 heterodimer) plus H9253H9280 and H9254H9280 heterodimers. The external domains of the H9253, H9254, and H9280 chains of CD3 are similar to the immunoglobulin fold, which facilitates their interaction with the T-cell receptor and each other. Ionic interactions also may occur between the oppositely charged transmembrane re- gions in the TCR and CD3 chains. The long cytoplasmic tails of the CD3 chains contain a common sequence, the immunoreceptor tyrosine-based activation motif (ITAM), which functions in signal transduction. NH 2 α γε β S S S S S S S S S S S S S S S S S S SS NH 2 + + + ? ? ? ? ? ? εδ 9 ζζ COOH COOH COOH (248) COOH (282) COOH COOH COOH COOH 143 ITAM 30 TCR NH 2 NH 2 NH 2 NH 2 90 105 116 130 130 160 185 185 106 150 105 80 22 90 23 91 134 184 140 201 222 255 8536d_ch09_200-220 8/2/02 9:49 AM Page 212 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: The H9253,H9254, and H9280chains of CD3 are members of the immuno- globulin superfamily, each containing an immunoglobulin- like extracellular domain followed by a transmembrane region and a cytoplasmic domain of more than 40 amino acids. The H9256 chain has a distinctly different structure, with a very short external region of only 9 amino acids, a trans- membrane region, and a long cytoplasmic tail containing 113 amino acids. The transmembrane region of all the CD3 polypeptide chains contains a negatively charged aspartic acid residue that interacts with one or two positively charged amino acids in the transmembrane region of each TCR chain. The cytoplasmic tails of the CD3 chains contain a motif called the immunoreceptor tyrosine-based activation mo- tif (ITAM). ITAMs are found in a number of other receptors, including the Ig-H9251/Ig-H9252 heterodimer of the B-cell receptor complex and the Fc receptors for IgE and IgG. The ITAM sites have been shown to interact with tyrosine kinases and to play an important role in signal transduction. In CD3, the H9253, H9254, and H9280 chains each contain a single copy of ITAM, whereas the H9256 and H9257 chains contain three copies (see Figure 9-9). The function of CD3 in signal transduction is described more fully in Chapter 10. T-Cell Accessory Membrane Molecules Although recognition of antigen-MHC complexes is medi- ated solely by the TCR-CD3 complex, various other mem- brane molecules play important accessory roles in antigen recognition and T-cell activation (Table 9-4). Some of these molecules strengthen the interaction between T cells and antigen-presenting cells or target cells, some act in signal transduction, and some do both. CD4 and CD8 Coreceptors Bind to Conserved Regions of MHC Class II or I Molecules. T cells can be subdivided into two populations according to their expression of CD4 or CD8 membrane molecules. As de- scribed in preceding chapters, CD4 H11001 T cells recognize anti- gen that is combined with class II MHC molecules and function largely as helper cells, whereas CD8 H11001 T cells recog- nize antigen that is combined with class I MHC molecules and function largely as cytotoxic cells. CD4 is a 55-kDa monomeric membrane glycoprotein that contains four ex- tracellular immunoglobulin-like domains (D 1 –D 4 ), a hy- drophobic transmembrane region, and a long cytoplasmic tail (Figure 9-10) containing three serine residues that can be phosphorylated. CD8 generally takes the form of a disulfide- linked H9251H9252 heterodimer or of an H9251H9251 homodimer. Both the H9251 and H9252 chains of CD8 are small glycoproteins of ap- proximately 30–38 kDa. Each chain consists of a single ex- tracellular immunoglobulin-like domain, a hydrophobic transmembrane region, and a cytoplasmic tail (Figure 9-10) containing 25–27 residues, several of which can be phos- phorylated. CD4 and CD8 are classified as coreceptors based on their abilities to recognize the peptide-MHC complex and their roles in signal transduction. The extracellular domains of CD4 and CD8 bind to the conserved regions of MHC mole- cules on antigen-presenting cells (APCs) or target cells. Crys- tallographic studies of a complex composed of the class I MHC molecule HLA-A2, an antigenic peptide, and a CD8 H9251H9251 homodimer indicate that CD8 binds to class I molecules T-Cell Receptor CHAPTER 9 213 TABLE 9-4 Selected T-cell accessory molecules FUNCTION Signal Member of Name Ligand Adhesion transduction Ig superfamily CD4 Class II MHC H11001H11001 H11001 CD8 Class I MHC H11001H11001 H11001 CD2 (LFA-2)CD58 (LFA-3) H11001H11001 H11001 LFA-1 (CD11a/CD18) ICAM-1 (CD54) H11001 ? H11001/(H11002) CD28 B7 ? H11001H11001 CTLA-4 B7 ? H11001H11002 CD45RCD22 H11001H11001 H11001 CD5 CD72 ? H11001H11002 8536d_ch09_200-220 8/22/02 2:51 PM Page 213 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: by contacting the MHC class I H92512 and H92513 domains as well as having some contact with H9252 2 -microglobulin (Figure 9-11a). The orientation of the class I H92513 domain changes slightly upon binding to CD8. This structure is consistent with a sin- gle MHC molecule binding to CD8; no evidence for the pos- sibility of multimeric class I–CD8 complexes was observed. Similar structural data document the mode by which CD4 binds to the class II molecule. The contact between CD4 and MHC II involves contact of the membrane-distal domain of CD4 with a hydrophobic pocket formed by residues from the H92512 and H92522 domains of MHC II. CD4 facilitates signal trans- duction and T-cell activation of cells recognizing class II– peptide complexes (Figure 9-11b). Whether there are differences between the roles played by the CD4 and CD8 coreceptors remains open to speculation. Despite the similarities in structure, recall that the nature of the binding of peptide to class I and class II molecules differs in that class I has a closed groove that binds a short peptide with a higher degree of specificity. Recent data shown below indicate that the angle at which the TCR approaches the pep- tide MHC complex differs between class I and II. The differ- ences in roles played by the CD4 and CD8 coreceptors may be due to these differences in binding requirements. As will be explained in Chapter 10, binding of the CD4 and CD8 molecules serves to transmit stimulatory signals to the T cells; the signal-transduction properties of both CD4 and 214 PART II Generation of B-Cell and T-Cell Responses βα S S S S S S CD8 D 3 D 4 S S D 2 S S CD4 D 1 S S FIGURE 9-10 General structure of the CD4 and CD8 coreceptors. CD8 may take the form of an H9251H9252 heterodimer, or an H9251H9251 homodimer. The monomeric CD4 molecule contains four Ig-fold domains; each chain in the CD8 molecule contains one. FIGURE 9-11 Interactions of coreceptors with TCR and MHC mol- ecules. (a) Ribbon diagram showing three-dimensional structure of an HLA-A2 MHC class I molecule bound to a CD8 H9251H9251 homodimer. The HLA-A2 heavy chain is shown in green, H9252 2 -microglobulin in gold, the CD8 H92511 in red, the CD8 H92512 in blue, and the bound peptide in white. A flexible loop of the H92513 domain (residues 223–229) is in con- tact with the two CD8 subunits. In this model, the right side of CD8 would be anchored in the T-cell membrane, and the lower left end of the class I MHC molecule (the H92513 domain) is attached to the surface of the target cell. (b) Interaction of CD4 with the class II MHC pep- tide complex (pMHCII). [Part (a) from Gao et al., 1997, Nature, 387:630; part (b) from Wang et al., 2001, PNAS, 98(19): 10799.] TCR pMHCII CD4 H92511 H92512 Class I MHC H92513 CD8 H9252 2 -microglobulin (a) (b) 8536d_ch09_200-220 8/23/02 12:01 PM Page 214 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: CD8 are mediated through their cytoplasmic domains. Re- cent data on the interaction between CD4 and the peptide– class II complex indicates that there is very weak affinity between them, suggesting that recruitment of molecules involved in signal transduction may be the major role for CD4. Affinity of TCR for Peptide-MHC Complexes Is Weak Compared with Antibody Binding The affinity of T-cell receptors for peptide-MHC complexes is low to moderate, with K d values ranging from 10 H110024 to 10 H110027 M. This level of affinity is weak compared with antigen-antibody interactions, which generally have K d val- ues ranging from 10 H110026 to 10 H1100210 M (Figure 9-12a). However, T-cell interactions do not depend solely on binding by the TCR; cell-adhesion molecules strengthen the bond between a T cell and an antigen-presenting cell or a target cell. Several accessory membrane molecules, including CD2, LFA-1, CD28, and CD45R bind independently to other ligands on antigen-presenting cells or target cells (see Table 9-4 and Figure 9-12b). Once cell-to-cell contact has been made by the adhesion molecules, the T-cell receptor may scan the membrane for peptide-MHC complexes. During activa- tion of a T cell by a particular peptide-MHC complex, there is a transient increase in the membrane expression of T-Cell Receptor CHAPTER 9 215 FIGURE 9-12 Role of coreceptors in TCR binding affinity. (a) Affinity constants for various biologic systems. (b) Schematic dia- gram of the interactions between the T-cell receptor and the pep- tide-MHC complex and of various accessory molecules with their ligands on an antigen-presenting cell (left) or target cell (right). Binding of the coreceptors CD4 and CD8 and the other accessory molecules to their ligands strengthens the bond between the inter- acting cells and/or facilitates the signal transduction that leads to activation of the T cell. Class I MHC LFA-3 CD45R CD22 ICAM-1LFA-1 CD8 T C cell Strong binding Affinity constant (mol/L) CD2 Antigen-presenting cell CD2 CD45R CD4 CD28 LFA-1 Class II MHC Peptide T H cell 10 –4 10 –5 10 –6 10 –7 10 –8 10 –9 10 –10 10 –11 TCR? CD3 S S S S S S CD22 B7 Antigen Peptide Antigen Target cell LFA-3 ICAM-1 TCR? CD3 T-cell receptors Adhesion Molecules Growth Factor Receptors Antibodies (a) (b) Weak 8536d_ch09_200-220 8/22/02 2:51 PM Page 215 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: 216 PART II Generation of B-Cell and T-Cell Responses FIGURE 9-13 Three-dimensional structures for the TCR-MHC- peptide complex. (a) Model showing the interaction between the human TCR (top, yellow) and the HLA-A2 class I MHC molecule (bottom, blue) with bound HTLV-I Tax peptide (white and red). (b) Backbone tube diagram of the ternary complex of mouse TCR bound to the class I MHC H-2K b molecule and peptide (green tube numbered P1–P8). CDR1 and 2 of the TCR H9251-chain variable domain (V H9251 ) are colored pink; CDR 1 and 2 of the H9252-chain variable domain (V H9252 ) are blue, and the CDR3s of both chains are green. The HV4 of the H9252 chain is orange. (c) MHC molecule viewed from above (i.e., from top of part (a), with the hypervariable loops (1–4) (a) TCR V H9252 V H9251 H92511 H92512 Peptide Class I MHC (c) V H9251 4 2 1 3 V H9252 2 1 3 4 C H9251 ( b) C H9252 V H9251 P1 P8 V H9252 H92513 H92512 H92511 H9252 2 m H92512 H92523 H92513 H92522 H92521 H92511 HV4 H9252 H9251 (d) of the human TCR H9251 (red) and H9252 (yellow) variable chains super- imposed on the Tax peptide (white) and the H92511 and H92512 domains of the HLA-A2 MHC class I molecule (blue). (d) CDR regions of mouse TCR H9251 and H9252 chains viewed from above, showing the sur- face that is involved in binding the MHC-peptide complex. The CDRs are labeled according to their origin (for example, H92511 is CDR1 from the H9251 chain). HV4 is the fourth hypervariable region of the H9252 chain. [Parts (a) and (c) from D. N. Garboczi et al., 1996, Nature 384:134–141, courtesy of D. C. Wiley, Harvard University; parts (b) and (d) from C. Garcia et al., 1996, Science 274:209, cour- tesy of C. Garcia, Scripps Research Institute.] 8536d_ch09_200-220 8/2/02 9:49 AM Page 216 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: T-Cell Receptor CHAPTER 9 217 cell-adhesion molecules, causing closer contact between the interacting cells, which allows cytokines or cytotoxic substances to be transferred more effectively. Soon after ac- tivation, the degree of adhesion declines and the T cell de- taches from the antigen-presenting cell or target cell. Like CD4 and CD8, some of these other molecules also function as signal-transducers. Their important role is demonstrated by the ability of monoclonal antibodies specific for the binding sites of the cell-adhesion molecules to block T-cell activation. Three-Dimensional Structures of TCR-Peptide-MHC Complexes The interaction between the T-cell receptor and an antigen bound to an MHC molecule is central to both humoral and cell-mediated responses. The molecular elements of this in- teraction have now been described in detail by x-ray crys- tallography for TCR molecules binding to peptide–MHC class I and class II complexes. A three-dimensional struc- ture has been determined for the trimolecular complex, in- cluding TCR H9251 and H9252 chains and an HLA-A2 molecule to which an antigenic peptide is bound. Separate studies de- scribe a mouse TCR molecule bound to peptides com- plexed with the mouse class I molecule H-2K b and with the mouse class II IA k molecule. The comparisons of the TCR complexed with either class I or class II suggest that there are differences in how the TCR contacts the MHC-peptide complex. Newly added to our library of TCR structures is that of a H9253H9254 receptor bound to an antigen that does not re- quire processing. From x-ray analysis, the TCR-peptide-MHC complex consists of a single TCR molecule bound to a single MHC molecule and its peptide. The TCR contacts the MHC mole- cule through the TCR variable domains (Figure 9-13 a,b). Although the structures of the constant region of the TCR H9251 chain and the MHC H92513 domain were not clearly established by studies of the crystallized human complexes (see Figure 9-13a), the overall area of contact and the structure of the complete TCR variable regions were clear. The constant re- gions were established by studies of the mouse complex, which showed the orientation proposed for the human models (see Figure 9-13b). Viewing the MHC molecule with its bound peptide from above, we can see that the TCR is sit- uated across it diagonally, relative to the long dimension of the peptide (Figure 9-13c). The CDR3 loops of the TCR H9251 and H9252 chains meet in the center of the peptide; and the CDR1 loop of the TCR H9251 chain is at the N terminus of the peptide, while CDR1 of the H9252 chain is at the C terminus of the peptide. The CDR2 loops are in contact with the MHC molecule; CDR2H9251 is over the H92512 domain alpha helix and CDR2H9252 over the H92511 domain alpha helix (Figure 9-13c). A space-filling model of the binding site viewed from above (looking down into the MHC cleft) indicates that the pep- tide is buried beneath the TCR and therefore is not seen from this angle (Figure 9-13d). The data also show that the fourth hypervariable regions of the H9251 and H9252 chains are not in contact with the antigenic peptide. As predicted from data for immunoglobulins, the recog- nition of the peptide-MHC complex occurs through the variable loops in the TCR structure. CDR1 and CDR3 from both the TCR H9251 and the TCR H9252 chain contact the peptide and a large area of the MHC molecule. The peptide is buried (see Figure 9-13d) more deeply in the MHC mole- cule than it is in the TCR, and the TCR molecule fits across the MHC molecule, contacting it through a flat surface of the TCR at the “high points” on the MHC molecule. The fact that the CDR1 region contacts both peptide and MHC suggests that regions other than CDR3 are involved in pep- tide binding. TCRs Interact Differently with Class I and Class II Molecules Can the conclusions drawn from the three-dimensional structure of TCR–peptide–class I complexes be extrapo- lated to interactions of TCR with class II complexes? Ellis Reinherz and his colleagues resolved this question by analy- sis of a TCR molecule in complex with a mouse class II mol- ecule and its specific antigen. While the structures of the peptide-binding regions in class I and class II molecules are similar, Chapter 7 showed that there are differences in how they accommodate bound peptide (see Figures 7-10a and b). A comparison of the interactions of a TCR with class I MHC–peptide and class II–peptide reveals a significant difference in the angle at which the TCR molecule sits on the MHC complexes (Figure 9-14). Also notable is a greater number of contact residues between TCR and class II MHC, which is consistent with the known higher affinity of interaction. However, it remains to be seen whether the ev- ident difference in the number of contact points will be true for all class I and II structures. (a) TCR–peptide–class I MHC (b) TCR–peptide–class II MHC FIGURE 9-14 Comparison of the interactions between H9251H9252 TCR and (a) class I MHC–peptide, and (b) class II MHC–peptide. The TCR (wire diagram) is red in (a), blue-green in (b); the MHC mole- cules are shown as surface models; peptide is shown as ball and stick. [From Reinherz et al., 1999, Science 286:1913.] 8536d_ch09_200-220 8/22/02 2:51 PM Page 217 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: Alloreactivity of T Cells The preceding sections have focused on the role of MHC molecules in the presentation of antigen to T cells and the in- teractions of TCRs with peptide-MHC complexes. However, as noted in Chapter 7, MHC molecules were first identified because of their role in rejection of foreign tissue. Graft- rejection reactions result from the direct response of T cells to MHC molecules, which function as histocompatibility antigens. Because of the extreme polymorphism of the MHC, most individuals of the same species have unique sets of MHC molecules, or histocompatibility antigens, and are considered to be allogeneic, a term used to describe geneti- cally different individuals of the same species (see Chapter 21). Therefore, T cells respond even to allografts (grafts from members of the same species), and MHC molecules are con- sidered alloantigens. Generally, CD4 H11001 T cells are alloreactive to class II alloantigens, and CD8 H11001 T cells respond to class I alloantigens. The alloreactivity of T cells is puzzling for two reasons. First, the ability of T cells to respond to allogeneic histocom- patibility antigens alone appears to contradict all the evi- dence indicating that T cells can respond only to foreign antigen plus self-MHC molecules. In responding to allo- geneic grafts, however, T cells recognize a foreign MHC mol- ecule directly. A second problem posed by the T-cell response to allogeneic MHC molecules is that the frequency of allore- active T cells is quite high; it has been estimated that 1%–5% of all T cells are reactive to a given alloantigen, which is higher than the normal frequency of T cells reactive with any particular foreign antigenic peptide plus self-MHC mole- cule. This high frequency of alloreactive T cells appears to contradict the basic tenet of clonal selection. If 1 T cell in 20 reacts with a given alloantigen and if one assumes there are on the order of 100 distinct H-2 haplotypes in mice, then there are not enough distinct T-cell specificities to cover all the unique H-2 alloantigens, let alone foreign antigens dis- played by self-MHC molecules. One possible and biologically satisfying explanation for the high frequency of alloreactive T cells is that a particular T-cell receptor specific for a foreign antigenic peptide plus a self-MHC molecule can also cross-react with certain allo- geneic MHC molecules. In other words, if an allogeneic MHC molecule plus allogeneic peptide structurally resem- bles a processed foreign peptide plus self-MHC molecule, the same T-cell receptor may recognize both peptide-MHC complexes. Since allogeneic cells express on the order of 10 5 class I MHC molecules per cell, T cells bearing low-affinity cross-reactive receptors might be able to bind by virtue of the high density of membrane alloantigen. Foreign antigen, on the other hand, would be sparsely displayed on the membrane of an antigen-presenting cell or altered self-cell associated with class I or class II MHC molecules, limiting responsiveness to only those T cells bearing high-affinity receptors. Information relevant to mechanisms for alloreactivity was gained by Reiser and colleagues, who determined the struc- ture of a mouse TCR complexed with an allogeneic class I molecule containing a bound octapeptide. This analysis re- vealed a structure similar to those reported for TCR bound to class I self-MHC complexes, leading the authors to conclude that allogeneic recognition is not unlike recognition of self- MHC antigens. The absence of negative selection for the pep- tides contained in the foreign MHC molecules can contribute to the high frequency of alloreactive T cells. This condition, coupled with the differences in the structure of the exposed portions of the allogeneic MHC molecule, may account for the phenomenon of alloreactivity. An explana- tion for the large number of alloreactive cells can be found in the large number of potential antigens provided by the for- eign molecule plus the possible peptide antigens bound by them. SUMMARY a73 Most T-cell receptors, unlike antibodies, do not react with soluble antigen but rather with processed antigen bound to a self-MHC molecule; certain H9253H9254 receptors recognize antigens not processed and presented with MHC. a73 T-cell receptors, first isolated by means of clonotypic mon- oclonal antibodies, are heterodimers consisting of an H9251 and H9252 chain or a H9253 and H9254 chain. a73 The membrane-bound T-cell receptor chains are orga- nized into variable and constant domains. TCR domains are similar to those of immunoglobulins and the V region has hypervariable regions. a73 TCR germ-line DNA is organized into multigene families corresponding to the H9251, H9252, H9253, and H9254 chains. Each family contains multiple gene segments. a73 The mechanisms that generate TCR diversity are generally similar to those that generate antibody diversity, although somatic mutation does not occur in TCR genes, as it does in immunoglobulin genes. a73 The T-cell receptor is closely associated with the CD3, a complex of polypeptide chains involved in signal transduction. a73 T cells express membrane molecules, including CD4, CD8, CD2, LFA-1, CD28, and CD45R, that play accessory roles in T-cell function or signal transduction. a73 Formation of the ternary complex TCR-antigen-MHC re- quires binding of a peptide to the MHC molecule and binding of the complex by the T-cell receptor. a73 Interactions between TCR and MHC class I/peptide differ from those with MHC class II/peptide in the contact points between the TCR and MHC molecules. a73 The H9253H9254 T-cell receptor is distinguished by ability to bind native antigens and by differences in the orientation of the variable and constant regions. 218 PART II Generation of B-Cell and T-Cell Responses 8536d_ch09_200-220 8/22/02 2:51 PM Page 218 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: a73 In addition to reaction with self MHC plus foreign anti- gens,T cells also respond to foreign MHC molecules, a re- action that leads to rejection of allogeneic grafts. References Allison, T. J., et al. 2001. Structure of a human H9253H9254 T-cell antigen receptor. Nature 411:820. Gao, G. F., et al. 1997. Crystal structure of the complex between human CD8H9251H9251 and HLA-A2. Nature 387:630. Garboczi, D. N., et al. 1996. Structure of the complex between human T-cell receptor, viral peptide, and HLA-A2. Nature 384:134. Garcia, K. C., et al. 1996. An H9251H9252 T-cell receptor structure at 2.5 ? and its orientation in the TCR-MHC complex. Science 274:209. Garcia, K. C., et al. 1998. T-cell receptor–peptide–MHC interac- tions: biological lessons from structural studies. Curr. Opinions in Biotech. 9:338. Hayday, A. 2000. H9253H9254 Cells: A right time and a right place for a conserved third way of protection. Ann. Rev. Immunol. 18:1975. Hennecke J., and D. C. Wiley 2001. T-cell receptor–MHC inter- actions up close. Cell 104:1. Kabelitz, D., et al. 2000. Antigen recognition by H9253H9254 T lympho- cytes. Int. Arch. Allergy Immunol. 122:1. Reinherz, E., et al. 1999. The crystal structure of a T-cell receptor in complex with peptide and MHC class II. Science 286:1913. Reiser, J-B., et al. 2000. Crystal structure of a T-cell receptor bound to an allogeneic MHC molecule. Nature Immunology 1:291. Sklar, J., et al. 1988. Applications of antigen-receptor gene re- arrangements to the diagnosis and characterization of lym- phoid neoplasms. Ann. Rev. Med. 39:315. Xiong, Y., et al. 2001. T-cell receptor binding to a pMHCII ligand is kinetically distinct from and independent of CD4. J. Biol. Chem. 276:5659. Zinkernagel, R. M., and P. C. Doherty. 1974. Immunological sur- veillance against altered self-components by sensitized T lym- phocytes in lymphocytic choriomeningitis. Nature 251:547. USEFUL WEB SITES http://imgt.cines.fr A comprehensive database of genetic information on TCRs, MHC molecules, and immunoglobulins, from the Interna- tional ImmunoGenetics Database, University of Montpelier, France. http://www.bioscience.org/knockout/tcrab.htm This location presents a brief summary of the effects of TCR knockouts. Study Questions CLINICAL FOCUS QUESTION A patient presents with an enlarged lymph node, and a T-cell lymphoma is suspected. However, DNA sampled from biopsied tissue shows no evidence of a predomi- nant gene rearrangement when probed with H9251 and H9252 TCR genes. What should be done next to rule out lymphocyte malignancy? 1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why. a. Monoclonal antibody specific for CD4 will coprecipitate the T-cell receptor along with CD4. b. Subtractive hybridization can be used to enrich for mRNA that is present in one cell type but absent in another cell type within the same species. c. Clonotypic monoclonal antibody was used to isolate the T-cell receptor. d. The T cell uses the same set of V, D, and J gene segments as the B cell but uses different C gene segments. e. The H9251H9252 TCR is bivalent and has two antigen-binding sites. f. Each H9251H9252T cell expresses only one H9252-chain and one H9251-chain allele. g. Mechanisms for generation of diversity of T-cell receptors are identical to those used by immunoglobulins. h. The Ig-H9251/Ig-H9252 heterodimer and CD3 serve analogous functions in the B-cell receptor and T-cell receptor, respectively. 2. What led Zinkernagel and Doherty to conclude that T-cell receptor recognition requires both antigen and MHC molecules? 3. Draw the basic structure of the H9251H9252 T-cell receptor and com- pare it with the basic structure of membrane-bound im- munoglobulin. 4. Several membrane molecules, in addition to the T-cell recep- tor, are involved in antigen recognition and T-cell activation. Describe the properties and distinct functions of the follow- ing T-cell membrane molecules: (a) CD3, (b) CD4 and CD8, and (c) CD2. 5. Indicate whether each of the properties listed below applies to the T-cell receptor (TCR), B-cell immunoglobulin (Ig), or both (TCR/Ig). a. ______ Is associated with CD3 b. ______ Is monovalent c. ______ Exists in membrane-bound and secreted forms d. ______ Contains domains with the immunoglobulin-fold structure e. ______ Is MHC restricted f. ______ Exhibits diversity generated by imprecise joining of gene segments g. ______ Exhibits diversity generated by somatic mutation 6. A major obstacle to identifying and cloning TCR genes is the low level of TCR mRNA in T cells. a. To overcome this obstacle, Hedrick and Davis made three important assumptions that proved to be correct. Describe each assumption and how it facilitated identification of the genes that encode the T-cell receptor. T-Cell Receptor CHAPTER 9 219 Go to www.whfreeman.com/immunology Self-Test Review and quiz of key terms 8536d_ch09_200-220 8/22/02 2:51 PM Page 219 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-: 8. Mice from different inbred strains listed in the left column of the accompanying table were infected with LCM virus. Spleen cells derived from these LCM-infected mice were then tested for their ability to lyse LCM-infected 51 Cr-labeled target cells from the strains listed across the top of the table. Indicate with (H11001) or (H11002) whether you would expect to see 51 Cr released from the labeled target cells. 9. The H9253H9254 T-cell receptor differs from the H9251H9252 in both structural and functional parameters. Describe how they are similar to one another and different from the B-cell antigen receptors. b. Suppose, instead, that Hedrick and Davis wanted to iden- tify the genes that encode IL-4. What changes in the three assumptions should they make? 7. Hedrick and Davis used the technique of subtractive hy- bridization to isolate cDNA clones that encode the T-cell re- ceptor. You wish to use this technique to isolate cDNA clones that encode several gene products and have available clones of various cell types to use as the source of cDNA or mRNA for hybridization. For each gene product listed in the left column of the table below, select the most appropriate. cDNA and mRNA source clones are from the following cell types: T H 1 cell line (A); T H 2 cell line (B); T C cell line (C); macrophage (D); IgA-secreting myeloma cell (E); IgG-secret- ing myeloma cell (F); myeloid progenitor cell (G); and B-cell line (H). More than one cell type may be correct in some cases. 220 PART II Generation of B-Cell and T-Cell Responses Gene product cDNA source mRNA source IL-2 CD8 J chain IL-1 CD3 Source Release of 51 Cr from of spleen LCM-infected target cells cells from LCM-infected B10.D2 B10 B10.BR (BALB/c H11003 B10) F 1 mice (H-2 d ) (H-2 b ) (H-2 k ) (H-2 b/d ) B10.D2 (H-2 d ) B10 (H-2 b ) BALB/c (H-2 d ) BALB/b (H-2 b ) 8536d_ch09_200-220 8/2/02 1:00 PM Page 220 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: