Ap and 78 TCell Receptors Structure and Roles

The domain structures of ap and 78 TCR heterodimers are strikingly similar to that of the immunoglobulins;

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 structurally 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 hypervariable regions, which appear to be equivalent to the complementarity determining regions (CDRs) in immunoglobulin light and heavy chains. There is an additional area of hypervariability (HV4) in the p chain that does not normally contact antigen and therefore is not considered a CDR.

In addition to the constant domain, each TCR chain contains 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 transmembrane region of 21 or 22 amino acids, which anchors each chain in the plasma membrane. The transmembrane domains of both chains are unusual in that they contain positively 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 contains a short cytoplasmic tail of 5-12 amino acids at the carboxyl-terminal end.

ap and yS T-cell receptors were initially difficult to investigate because, like all transmembrane proteins, they are insoluble. This problem was circumvented by expressing modified forms of the protein in vitro that had been engineered to contain premature in-frame stop codons that preclude translation of the membrane-binding sequence that makes the molecule insoluble.

The majority of T cells in the human and the mouse express T-cell receptors encoded by the ap genes. These receptors 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 yS cells react with antigen that is neither processed nor presented in the context of a MHC molecules.

Differences in the antigen-binding regions of ap and yS were expected because of the different antigens they recognize, but no extreme dissimilarities were expected. However, the recently completed three-dimensional structure for a yS receptor that reacts with a phosphoantigen, reported by Allison, Garboczi, and their coworkers, reveals significant

B-cell mIgM

B-cell mIgM

nh2

Connecting sequence Transmembrane region (Tm)

Cytoplasmic tail (CT)

ap T-cell receptor a-chain P-chain NH2 NH2

Connecting sequence Transmembrane region (Tm)

Cytoplasmic tail (CT)

-S-S-

+ +

8 o

COOH (282)

COOH (248)

COOH (282)

FIGURE 9-3

Schematic diagram illustrating the structural similarity between the ap T-cell receptor and membrane-bound IgM on B cells. The TCR a and p chain each contains two domains with the im-munoglobulin-fold structure. The ammo-terminal domains (Va and Vp) exhibit sequence variation and contain three hypervariable regions equivalent to the CDRs in antibodies. The sequence of the constant domains (Ca and Cp) 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^ region. TCR molecules interact with CD3 via positively charged amino acid residues (indicated by +) in their transmembrane regions. Numbers indicate the length of the chains in the TCR molecule. Unlike the antibody molecule, which is bivalent, the TCR is monovalent.

p differences in the overall structures of the two receptor types, pointing to possible functional variation. The receptor they studied was composed of the 79 and 82 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 78 receptor is specific. This antigen is recognized without MHC presentation.

The most striking feature of the structure is how it differs from the ap receptor in the orientation of its V and C regions. The so-called elbow angle between the long axes of the V and C regions of 78 TCR is 111°; in the ap 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 mechanisms and in how the molecules interact with coreceptor molecules.

The number of 78 cells in circulation is small compared with cells that have ap receptors, and the V gene segments of 78 receptors exhibit limited diversity. As seen from the data in Table 9-1, the majority of 78 cells are negative for both CD4 and CD8, and most express a single 78-chain subtype. In humans the predominant receptor expressed on circulating 78 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 78 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 78 cells in the mouse and of other species studied does not parallel that of humans, suggesting that the 78 response may be directed against pathogens commonly encountered by a given species. Furthermore, data indicating that 78 cells can secrete a spectrum of cytokines suggest that they may play a regulatory role in recruiting ap T cells to the site of invasion by pathogens. The recruited ap T cells would presumably display a broad spectrum of receptors; those with the highest

Comparison of ap and 78 T cells

FIGURE 9-4

Comparison of the 78 TCR and ap TCR. The difference in the elbow angle is highlighted with black lines. [From T. Allison et al., 2001, Nature 411:820.]

FIGURE 9-4

Comparison of the 78 TCR and ap TCR. The difference in the elbow angle is highlighted with black lines. [From T. Allison et al., 2001, Nature 411:820.]

Feature ap T cells

78 T cells

Proportion of CD34 cells

TCR V gene germline repertoire

CD4/CD8 phenotype

CD4+

CD8+

CD4+CD8+

CD4-CD8-

MHC restriction

Ligands

90-99% Large

CD4+: MHC class II

CD8+: MHC class I

Peptide + MHC

No MHC restriction

Phospholipid antigen

SOURCE: D. Kabelitz et al., 1999, Springer Seminars in Immunopathology 21:55, p. 36.

affinity would be selectively activated and amplified to deal with the pathogen.

Organization and Rearrangement of TCR Genes

The genes that encode the ap and 78 T-cell receptors are expressed only in cells of the T-cell lineage. The four TCR loci (a,p, 7, and 8) 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 rearrangements of V and J segments in the a-chain and 7-chain families and V, D, and J segments in the p-chain and 8-chain families. In the mouse, the a-, p-, and 7-chain gene segments are located on chromosomes 14, 6, and 13, respectively. The 8-gene segments are located on chromosome 14 between the Va and Ja segments. The location of the 8-chain gene family is significant: a productive rearrangement of the a-chain gene segments deletes C8, so that, in a given T cell, the ap TCR receptor cannot be coexpressed with the 78 receptor.

Mouse germ-line DNA contains about 100 Va and 50 Ja gene segments and a single Ca segment. The 8-chain gene family contains about 10 V gene segments, which are largely distinct from the Va gene segments, although some sharing

Mouse TCR a-chain and S-chain DNA (chromosome 14)

LVa1 LVa2 L Van L Vs1 L Vsn Ds1Ds2Js1Js2 Cs L Vs5 Ja1Ja2Ja3 Jan Ca

Mouse TCR p-chain DNA (chromosome 6) (Vp n = 20 - 30)

LVp1 LVp2 LVpn Dp1 -Jp1.1-Jp1.7- Cp1 Dp2 -Jp2.1-Jp2.7- Cp2 LVp14

Mouse TCR y-chain DNA (chromosome 13)

L Vy5 L Vy2 L Vy4 L Vy3 Jy1 Cy1 L Jy3 Cy3 Cy2 Jy2 L L Jy4 Cy4

0 = Enhancer V = pseudogene

Vy1.3

Vy1.2 Vy1.1

FIGURE 9-5

Germ-line organization of the mouse TCR a-, p-, 7-, the various gene segments differs in some cases (see Table 9-2). and 8-chain gene segments. Each C gene segment is composed of a [Adaptedfrom D. Raulet, 1989, Annu. Rev. Immunol. 7:175, and M.

series of exons and introns, which are not shown. The organization Davis, 1990, Annu. Rev. Biochem. 59:475.] of TCR gene segments in humans is similar, although the number of of V segments has been observed in rearranged a- and 8-chain genes. Two D8 and two J8 gene segments and one C8 segment have also been identified. The p-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 Dp, six Jp, and one Cp. The 7-chain gene family consists of seven V7 segments and three different functional J7-C7 repeats. The organization of the TCR multigene families in humans is generally similar to that in mice, although the number of segments differs (Table 9-2).

TABLE 9-2

] TCR Multigene fami

lies

in humans

Chromosome location

NO. OF GENE SEGMENTS

Gene

V

D

JC

a Chain

14

50

70 1

8 Chain"

14

3

3

3 1

P Chain1

7

57

2

13 2

7 Chain*

7

14

5 2

"The 8-chain gene segments are located between the Va and Ja segments. 'There are two repeats, each containing 1 Dp, 6 or 7 Jp, and 1 Cp. 'There are two repeats, each containing 2 or 3 J7 and 1 C7. SOURCE: Data from P. A. H. Moss et al., 1992, Annu. Rev. Immunol. 10:71.

"The 8-chain gene segments are located between the Va and Ja segments. 'There are two repeats, each containing 1 Dp, 6 or 7 Jp, and 1 Cp. 'There are two repeats, each containing 2 or 3 J7 and 1 C7. SOURCE: Data from P. A. H. Moss et al., 1992, Annu. Rev. Immunol. 10:71.

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