Tcell

cancers, which include leukemia and lymphoma, involve the uncontrolled proliferation of a clonal population of T cells. Successful treatment requires quick and certain diagnosis in order to apply the most effective treatment. Once treatment is initiated, reliable tests are needed to determine whether the treatment regimen was successful. In principle, because T-cell cancers are clonal in nature, the cell population that is cancerous could be identified and monitored by the expression of its unique T-cell receptor molecules. However, this approach is rarely practical because detection of a specific TCR molecule requires the tedious and lengthy preparation of a specific antibody directed against its variable region (an anti-idiotype antibody). Also, surface expression of the TCR molecule occurs somewhat late in the development ofthe 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 protein products. The pattern resulting from rearrangement of the TCR genes can provide a unique marker for the cancerous T cell. Because rearrangement of the TCR genes in the T cells occurs before the product molecule is expressed, T cells in early stages of development can be detected. The unique gene fragments that result from TCR gene rearrangement can be detected by simple molecular-biological techniques and provide a true fingerprint for a clonal cell population.

DNA patterns that result from rearrangement of the genes in the TCR p region are used most frequently as markers. There are approximately 50 Vp gene segments that can rearrange to one of two D-region gene segments and subsequently 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 p complex is known, the appropriate probes and restriction enzymes 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 persist; 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 variety 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 persistent enlargement of the nodes represents 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 observed enlargement was due to the cancerous 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 rearrange 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 technique, 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 proliferation 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 ofits developmental stage or because it is of another lineage (78 T cells, for example).

If the DNA fragment test and other diagnostic criteria indicate that the patient has a lymphoid cell cancer, treatment by

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 Va and 50 Ja gene segments can generate 5 X 103 possible VJ combinations for the TCR a chain. Similarly, 25 Vp, 2 Dp, and 12 Jp gene segments can give 6 X 102 possible combinations. Although there are fewer TCR Va and Vp gene segments than immunoglobulin VH and Va 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 X 103 Va combinations with 6 X 102 Vp combinations can generate 3 X 106 possible combinations

si i

L Vpl

L Vpl

-coRI 42 kb -coRI

L Vpl

-coRI 42 kb -coRI

L Vpl

Southern blot probed with C DNA

Digestion of human TCR P-chain DNA in a germ-line (nonrearranged) configuration with EcoRI and then probing with a C-region sequence will detect the indicated C-containing fragments by Southern blotting. When the DNA has rearranged, a 5' restriction site will be excised. Digestion with EcoRI will yield a different fragment unique to the specific Vp and Jp 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 p 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 5' primer (red bar) is based on a unique sequence in the (Vp) gene segment used by the cancerous clone (Vp2 in this example) and the 3' 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.

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 regimen is successful, the number of cancerous 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 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 p-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). Recently, quantitative PCR methods have been used to follow patients who are in remission in order to make decisions about resuming treatment if the number of cancerous cells, as estimated by these techniques, has risen above a certain level. Therefore, the presence of the rearranged DNA in the clonal population of T cells gives the clinician a valuable tool for diagnosing lymphoid-cell cancer and for monitoring the progress of treatment.

for the ap T-cell receptor. Additional means to generate diversity in the TCR V genes are described below, so 3 X 106 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 sequences (RSSs) in TCR p- and 8-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 joining rule is observed. Thus, it is possible for a Vp gene segment to join directly with a Jp or a Dp gene segment, generating a (VJ)p or (VDJ)p unit.

Alternative joining of h-chain gene segments generates similar units; in addition, one D8 can join with another,

Sources of possible diversity in mouse immunoglobulin and TCR genes

IMMUNOGLOBULINS

«P T-CELL RECEPTOR

78 T-CELL RECEPTOR

Mechanism of diversity

H Chain k Chain

Chain

P Chain

7 Chain

8 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 X 13 X 4

85 X 4

100 X 50

25 X 2 X 12

7X3

10 X 2 X

and V-D-J joining

= 7 X 103

= 3.4 X 102

= 5 X 103

= 6 X 102

= 21

= 40

Alternative joining

-

-

-

+

-

+

of D gene segments

(some)

(often)

Junctional flexibility

+

+

+

+

+

+

N-region nucleotide addition''

+

-

+

+

+

+

P-region nucleotide addition

+

+

+

+

+

+

Somatic mutation

+

+

-

-

-

-

Combinatorial

association of chains

+

+

+

*A plus sign (+) indicates mechanism

makes a significant i

contribution to diversity but to

an unknown extent.

A minus sign (—) indicates mechanism does not operate.

'See Figure 9-8d for theoretical number of combinations generated by N-region addition.

A minus sign (—) indicates mechanism does not operate.

'See Figure 9-8d for theoretical number of combinations generated by N-region addition.

yielding (VDDJ)8 and, in humans, (VDDDJ)8. This mechanism, which cannot operate in Ig heavy-chain DNA, generates considerable additional diversity in TCR genes.

The joining of gene segments during TCR-gene rearrangement exhibits junctional flexibility. As with the Ig genes, this flexibility can generate many nonproductive rearrangements, but it also increases diversity by encoding several 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 rearrangement (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 diversity. 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 junction, generating up to 5461 possible combinations, assuming random selection of nucleotides (see Figure 9-8d). Some of these combinations, however, lead to nonproductive rearrangements by inserting in-frame stop codons that prematurely 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 generate as many as 1013 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 number 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 segments 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 a helices of MHC molecules. Although this is an attractive idea, it is

T-CELL RECEPTOR

(a) Combinatorial V-J and V-D-J joining V DJ

ß and S chains a and y chains

IMMUNOGLOBULIN

V DJ

H chain

L chain

(b) Alternative joining of D gene segments L vs ds ds js

= One-turn

RSS = Two-turn RSS

L vh dh jh

i vh-dh-jh only

(c) Junctional flexibility

One-turn RSS DS

(c) Junctional flexibility

One-turn RSS DS

CACTGTG

GTGGACT

GATGCTCC

CACAGTG

Two-turn RSS

Two-turn RSS

One-turn RSS

DH

CACTGTG

ATGGACT

TGGCCG

CACAGTG

VH

Two-turn RSS

(d) N-region nucleotide addition VJ

T = Addition of

0-6 nucleotides (5461 permutations)

V DJ

Heavy chain

s chain

FIGURE 9-8

Comparison of mechanisms for generating diversity in TCR genes and immunoglobulin genes. In addition to the mechanisms 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.

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 undergo 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 specificity does not change after thymic selection and therefore 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.

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