A Particular TCR β Variable Region Used by
T Cells Infiltrating Kidney Transplants
Christophe Baron, David H. Sachs and Christian LeGuern
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References
J Immunol 2001; 166:2589-2596; ;
doi: 10.4049/jimmunol.166.4.2589
http://www.jimmunol.org/content/166/4/2589
A Particular TCR ␤ Variable Region Used by T Cells
Infiltrating Kidney Transplants1
Christophe Baron, David H. Sachs, and Christian LeGuern2
M
iniature swine have been used extensively as models
for human allogeneic organ transplantation (reviewed
in Ref. 1). The availability of strains of miniature
swine MHC homozygous, as well as MHC recombinant, has permitted dissection of the respective contributions of class I and II
Ags in graft rejection. Without the adjunct of an immunosuppressive treatment, a two-haplotype MHC class I disparity leads to
kidney graft rejection in this model (2). However, treatment with
cyclosporine for 12 days, beginning on the day of transplantation,
uniformly promotes specific tolerance to such grafts (2). Rejection
of class I-disparate kidney grafts is associated with infiltrating cytotoxic T cells, whereas drug-induced tolerance appears to use peripheral mechanisms involving regulatory T cells (3). Similar T
cell subsets have been described in other transplantation models
(4), although only sparse information is available on their fine
specificities and functions in the rejection and/or tolerance process.
To characterize the fine specificity of the TCR of the various T cell
subsets involved in our renal transplant model, we have developed
and tested the molecular tools for analyzing the complementaritydetermining region 3 (CDR3)3 length polymorphism of the porcine
TCR ␤ segments (5). To this end, we first established the nucleotide sequences of 19 functional porcine V␤ segments, among
which we identified a new V␤100 segment. Twelve J␤ along with
two D␤ sequences were also described. This set of porcine se-
Transplantation Biology Research Center, Massachusetts General Hospital/Harvard
Medical School, Boston, MA 02129
Received for publication July 28, 2000. Accepted for publication November 21, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Heath Grants 2RO1
AI33053, 2RO1 AI31046, and 2PO1 HL18646. C.B. was supported by grants from
“Ministere des affaires etrangeres bourse Lavoisier” and from “Societe Francaise De
Nephrologie.”
2
Address correspondence and reprint requests to Dr. Christian Leguern, Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Building 149-9019, 13th Street, Boston, MA 02129. E-mail address: leguern@helix.
mgh.harvard.edu
3
Abbreviation used in this paper: CDR3, complementarity-determining region 3.
Copyright © 2001 by The American Association of Immunologists
quences, along with the human and rodent V␤ sequences, represents the three most extensive V␤ collections described so far, as
well as an invaluable material for TCR repertoire studies in a clinically relevant model. In addition, pilot spectratyping studies for
V␤ usage demonstrated the dominance of V␤100⫹ cells in the
intragraft subset of lymphocytes.
Materials and Methods
RT-PCR analysis
All the pig V␤, J␤, and D␤ were obtained by PCR amplification followed
by cloning. PCR procedures were performed with RNA derived from 106
PBMC from normal miniature swine according to standard procedure (6).
The RNA was finally purified through a 5.7 M CsCl cushion in 25 mM
sodium citrate, and first strand cDNA was synthesized using 1 ␮g of RNA,
the Superscript reverse transcriptase (Life Technologies, Grand Island,
NY), and a poly d(T) primer (Life Technologies) according to the manufacturer’s recommendations. The resulting cDNA was amplified with the
C␤ primer (Table I) derived from the porcine C␤ sequence (7) in combination with either the PAN-1 V␤ primer or the PAN-2 V␤ primers (Table
I), each derived from a highly conserved region of human V␤ sequences
encompassing residue 98 –115 according to Kabat numbering (8).
The porcine V␤ segments, not detected in the first run of screening with
the pan V␤ primers, were amplified from a second set of degenerate V␤
oligonucleotides specific for a single V␤ subfamily. Two V␤-specific degenerate primers called V␤2 and V␤14 were designed to anneal to sequence stretches conserved among human, rat, cattle, and mouse V␤2 or
V␤14 (Table I). They were used following the RT-PCR conditions described above. 5⬘ truncated V␤ sequences generated from these two experimental approaches were completed by 5⬘ rapid amplifications of cDNA end (Life Technologies) (9) according to the manufacturer’s
recommendations. Alternatively, some V␤ sequences were cloned following RT-PCR amplification with C␤ and V␤ primers corresponding to the
peptide signal region (Table I). PCR amplification conditions were as follows: denaturation at 94°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 50 s. Thirty cycles were performed and terminated by
a 10-min extension time at 72°C.
Sequences analysis
The RT-PCR amplified products were digested with EcoRI and BglII restriction enzymes and electrophoresed on a 2% agarose gel. cDNA bands
of the expected size were excised from the gel, purified, and cloned into the
pBluescript KS⫹ plasmid (Stratagene, La Jolla, CA). Nucleotide sequences
of cloned V␤ and J␤ fragments were obtained in both directions using a
dideoxynucleotide termination reaction kit (Thermosequenase USB). Some
0022-1767/01/$02.00
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Immune tolerance to MHC class II identical renal grafts is achievable in miniature swine following a short immunosuppressive
treatment. Like in clinical transplants, swine-accepted allografts are primarily infiltrated by CD8ⴙ T cells, which are noncytotoxic
to the renal tissue. However, the actual specificity and function of these intragraft-infiltrating lymphocytes remain poorly understood. To develop the molecular tools to study TCR-associated functions of graft-infiltrating cells in a preclinical transplantation
model, we have determined the nucleotide sequence of 19 pig V␤, 12 J␤, and two D␤. Sequence comparisons identified 17 different
V␤ families and two J␤ clusters homologous to the human J␤1 and J␤2. The fact that the pig J␤1 segments were always found
joined to the D␤1-like sequence in numerous rearranged TCR ␤ cDNA suggests the existence of two D-J clusters in swine. These
results also imply that the polymorphism of the porcine TCR ␤ segments is similar to that found in human. Finally, we report the
discovery of a new and functional V␤ subfamily named V␤100, which exhibited similarity to the murine V␤2 sequence but had
no described V␤ homolog in humans. Pilot spectratyping studies on V␤ usage revealed a clonal dominance of V␤100ⴙ T cell
subsets among infiltrating cells in two accepted grafts. The Journal of Immunology, 2001, 166: 2589 –2596.
TCR ␤ DIVERSITY IN A PRECLINICAL MODEL OF ORGAN TRANSPLANTATION
2590
Table I. List of the V␤ and C␤ polynucleotide primers used in this study
Primers
Pan V␤ primers:
PAN-1 V␤
PAN-2 V␤
V␤-specific primers
BV14
BV2
C␤ primer
Signal peptide-derived primers
SP1
SP2
SP3
Pig V␤-specific primers
BV7
BV20
BV22
BV24
BV100
Sequence
5⬘-GGGAATTC-T-G/T-T-A/G/T-CTGGTA-C/T-C-A/G-A/G-CAG-3⬘
GGGAATTCT-G/T-T-A/C/T-C/T-TGGTA-C/T-C/A-A/G-A/G-CA
GGAATTCCCTGA-A/G-GGGTA-C/T-A/C-A-A/C-GTCTCT
GGAATTCGCCGT-G/T-C-A/C-A/G-TGGACTTTCA
GGGGATCCTCCGTGAGCCCATAGAAC
C-A/G-G-A/G-CTCCTCTGCTGTGTGG
GG-G/C-CGCTCTCCTTTCTCTG
A-C/G-TCTTCTGCTC-A/C-TTCTCCT
ACCTGTAACTACGAAGACCGC
CATCCTGAGTTCTACGAAGC
CGGCACGTACCTGACTCT
GACACCTCGGAAAACTTCAAA
TTCCTGGAGCAGATTATCA
RT-PCR spectratyping
RNA templates for spectratyping were purified from kidney biopsy and
PBL of miniature swine 11574 and 11560 tolerant to MHC class I disparate
renal grafts. Spectratyping for CDR3 length polymorphism was conducted
as described (10) with the following modifications. Specific amplifications
of V␤ 7, 20, 22, 24, and 100 transcripts were performed with pig V␤
specific primers (Table I) together with the antisense 5⬘-TCCGTGAGCCCATAGAACTG-3⬘ C␤ primer. Conditions for RT-PCR were as follows:
first strand cDNA corresponding to 0.1 ␮g of total RNA was amplified in
25 ␮l final volume containing 0.2 mM of each dNTP, 1.5 mM MgCl2, 50
mM KCl, 10 mM Tris-HCl (pH 9), 0.8 ␮M of each primer, and 0.5 U of
the Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA). Thirty cycles
were performed and comprised a denaturation at 94°C for 30 s, annealing
at 58°C for 40 s, and extension at 72°C for 50 s. Terminal extension was
for 10 min at 72°C.
Amplified products were then revealed on a sequencing gel following
two cycles of primer extension with a 32P-radiolabeled primer (5⬘-ATCTCCGCTTCCGATGGTTCAA-3⬘) annealing to the 5⬘ end of the C␤ region.
Spectratyping band profiles were then quantified by computer scanning
analysis using Molecular Analyst software (Bio-Rad, Richmond, CA).
Results
Pig V␤ nucleotide sequences
Three strategies were used to PCR amplify and clone the rearranged V␤ sequences from PBL cDNA of the MGH miniature
swine. A first set of pig sequences was obtained by using the
PAN-1 or PAN-2 V␤ primers together with the primer for C␤
(Table I). This approach led to the identification of 10 5⬘ truncated
pig V␤ sequences: V␤1, 6.1, 6.2, 7, 8, 12, 20, 21, 22.1, and 100.
The use of V␤-specific degenerate primers for the human V␤2 and
V␤14 (Table I) allowed amplification of their porcine counterparts.
The missing 5⬘ portion from all of these V␤ sequences was obtained by 5⬘ rapid amplification of cDNA ends (see Materials and
Methods). Finally, the full-length V␤4, V␤5, V␤10, V␤11, V␤17,
V␤22.2, and V␤24 sequences were cloned from RT-PCR amplifications with degenerate signal-peptide primers (Table I).
The nucleotide sequences of 19 open reading frames corresponding to the pig V␤ are presented in Fig. 1. The overall nucleotide homology between sequences ranged from 23 to 70%
(mean ⫽ 40%), similar to that found for human and mouse V␤
(reviewed in Ref. 11). For convenience, we have adopted the human V␤ nomenclature to designate the porcine counterparts with
high homology, as shown in Table II. Based on this nomenclature
(12, 13), pair comparison of nucleotide sequences using the Clustal
algorithm facilitated the assignment of the pig sequences into different V␤ families. Of the 19 sequences, 15 segregated into different single member V␤ families, sharing ⬍75% homology (Table III). Two additional families, V␤6 and V␤22, each contained
two distinct V␤ members, which displayed 90 and 93% sequence
homology, respectively (Table III). Finally, among the 15 single
member families, the open reading frame for the pig V␤100, although encoding for all of the critical structural residues of a V␤chain (Fig. 2), displayed only an average of 27% homology with
the other pig V␤ sequences of our panel, and had no corresponding
sequence in humans (Table II).
Porcine predicted V␤ amino acid sequences
Comparisons among the 19 predicted V␤ amino acid sequences
indicated that residues crucial for the integrity of the TCR threedimensional structure, such as 23Cys and 91Cys, were conserved in
the pig sequences to form the Ig-like V domain of the TCR ␤-chain
(Fig. 2). Other residues involved in ␤-chain contacts, such as
35
Tyr, 37Gln, and 92Ala, were conserved in 85% of cases. The
90
Tyr was replaced by a Leu in the pig V␤100, similar to what is
found in the mouse V␤2 counterpart (14). Most of the porcine V␤
sequences segregated into the two V␤ subgroups defined in other
species (8). Thus, the pig V␤1, 2, 4, 5, 6, 7, 8, 10, 21, 22, and 24
sequences clearly belonged to subgroup I, which has been defined
by invariant 65Phe and 86Asp residues that form a salt bridge with
64
Arg (8). Similarly, the V␤12, 14, 17, and 100 sequences belonged to subgroup II, which is characterized by a 65Tyr and no
Asp in position 86 (13). However, the representative features defining these subgroups were not found in the pig V␤11 and V␤20
amino acid sequences (Fig. 2).
The COOH-terminal sequence of the pig V␤ segments was deduced on the basis of the presence of the CASS consensus sequence found at the end of the majority of other species V␤ sequences. The substitution Ser 3 Arg in the CASR sequence of pig
V␤12 (Fig. 2) is likely to be the result of the V-J joining process,
because the sequence of another independently cloned V␤12
cDNA (clone 6, Fig. 3) also predicted a CASS sequence in that
region.
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sequencing was also performed at the Massachusetts General Hospital Department of Molecular Biology in the Sequencing Core Facility, which uses
a fluorescently labeled dideoxynucleotide chain termination method (Taq
DyeDeoxy Terminator cycle sequencing kit; Applied Biosystems, Foster
City, CA). The DNA samples were resolved by gel electrophoresis on an
ABI 377 PRISM automated sequencer. Sequence analysis and alignments
were performed using the Lasergene software (DNAstar, Madison, WI).
Sequence phylogenic analyses were performed by the Cluster method.
2591
The Journal of Immunology
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TCR ␤ DIVERSITY IN A PRECLINICAL MODEL OF ORGAN TRANSPLANTATION
2592
Table II. Percentages of homology between the human and pig V␤ sequences
Pig V␤ Subfamily
1
2
4
5
6
7
8
10
11
12
14
17
20
21
22
24
100
a
World Health Organization–International BV1 BV2 BV4 BV5 BV6 BV7 BV8 BV10 BV11 BV12 BV14 BV17 BV20 BV21 BV22 BV24 –
Union of Immunological Societies
TCR Nomenclature
Nucleotide homology (%)
78 71 78 71 72 70 78 68
73
65
80
79
77
71
62
74
–
Aminoacid homology (%)
70 61 70 67 62 63 71 52
72
57
67
74
71
61
52
68
–
a
Pig V␤ refer to TCRBV according to the World Health Organization nomenclature.
Table III. Percents homology among pig V␤ subfamiliesa
6.1
6.1
6.2
21
22.1
22.2
8
10
7
24
1
5
4
2
17
12
14
100
11
20
81
48
50
46
53
35
34
35
44
45
27
23
27
31
27
18
30
25
6.2
21
22.1
22.2
8
10
7
24
1
5
4
2
17
12
14
100
11
20
90
55
54
52
53
51
52
50
51
93
57
52
49
52
51
44
43
46
40
41
44
40
37
31
39
41
36
35
39
37
42
37
38
40
35
39
50
46
37
46
47
40
39
38
43
48
44
36
47
48
45
43
38
42
72
29
29
27
30
30
34
22
26
36
28
27
28
28
24
25
28
26
22
26
28
30
29
55
32
35
33
35
36
34
30
35
42
41
40
32
29
36
38
32
33
33
32
32
31
41
38
36
30
30
51
36
34
29
35
36
34
29
37
41
39
37
28
30
54
54
23
25
28
25
26
27
23
23
27
32
27
28
27
29
30
30
38
33
30
29
30
37
30
35
35
36
33
24
26
45
53
53
27
30
31
32
25
28
30
21
25
31
28
29
24
28
28
27
30
28
29
50
47
45
50
34
29
29
37
32
27
24
22
30
26
16
24
23
43
42
50
30
22
31
26
26
23
16
27
26
24
21
19
23
88
46
31
25
29
41
45
21
18
27
23
28
19
22
17
46
31
27
29
40
41
20
20
26
23
24
18
22
18
33
27
29
37
41
32
22
29
25
22
18
26
19
24
29
30
29
14
11
21
20
19
14
24
15
36
28
34
20
20
28
28
25
13
30
16
34
38
28
24
34
38
35
19
32
23
68
20
22
32
25
33
23
32
23
22
23
35
29
29
21
31
24
45
24
21
20
19
21
16
22
21
22
20
19
17
43
53
19
43
26
47
18
42
22
17
46
21
21
20
20
Pig V␤ subfamily names are shown on the x- and y-axes in boldface. The aminoacid and nucleotide sequence similarities are indicated in the lower and upper triangles,
respectively. x-axis ⫽ protein sequence homology; y-axis ⫽ nucleic sequence homology.
a
Characterization of pig TCR J␤ nucleotide sequences
Thirty-nine J␤ clones were fully analyzed, defining 12 distinct
predicted J␤ nucleotide sequences. As shown in Fig. 3, each J␤
sequence except J␤1.5 was found at least twice in independent
cDNA clones. The J-C boundaries were defined with respect to the
known sequence of the NH2-terminal portion of the pig C␤ (7).
The 11 deduced J␤ amino acid sequences ended with the consensus residues defined by Kimura et al. (15), such as 109Tyr and
111
Leu or Val or Tyr in position 111, which are essential to the
V␣/V␤ and V␤/C␤ interactions, respectively. The motif TTC/TGGN-NNN-GGN (FGXG), located within the core section of the
J␤, was invariant in pigs as it is in humans and mice (11, 15). Due
to the variability of the N region length and sequence, and to the
lack of detailed information on the porcine J and D clusters in
germline configuration, we were unable to determine the precise
D-J junction for each of the cloned J sequences. Nevertheless, the
basic contribution of germline J␤ sequences to the rearranged
CDR3 regions can be deduced, in part, by comparing the junctional sequences of the same J␤ in various TCR␤ cDNA clones
(Fig. 3). Such analysis led to the identification of 11 J␤ consensus
sequences, which varied in length from 42 to 48 nucleotides (Fig.
4). Because the pig J␤ 1.5 was found only in clone 15 (Fig. 3), its
predicted 5⬘ termini sequence was deduced from the strong homology with the human J␤ 1.5 (Fig. 4). Similarly to the V␤ designation, the pig J␤ nomenclature was deduced from the best ho-
mology scores observed between the pig and the corresponding
human J␤ sequences (Fig. 4).
Identification of a pig D␤ cluster
During the process of V␤ cDNA cloning, a cloned PCR product
was identified as a genomic DNA sequence, which contained a
TCR␤ D region in germline configuration. This D region was further characterized by the presence of conserved heptamer and
nonamer recombination signals separated by correct size spacer
(Fig. 5). In addition, the region located between the two nonamer
recombination signals showed 89% homology to the human D␤2
corresponding region, and only 50% to the human D␤1 region,
indicating that this porcine D region was likely the homolog of the
human genomic D␤2 cluster.
The high length variability of the sequence, located between the
V␤ and J␤ sections in the pig TCR␤ cDNA (Fig. 3), suggested the
contribution of N regions in the CDR3 polymorphism, as has been
observed in all other TCR␤ variable domains (16).
Clonal dominance among V␤100⫹ T lymphocytes within the
intragraft cell pool
Because the V␤100 sequence appeared to be absent in the human
T cell repertoire, we decided to ascertain whether swine T cell
clones using this particular segment could be found in clinically
relevant situations in this animal model. A biopsy of renal tissue
FIGURE 1. Nucleotide sequence alignment of 19 porcine V␤. Gaps were introduced to favor maximum alignment and are marked by hyphens.
Numbering corresponds to that of the mature ␤-chain as described for humans (Table II, Ref. 8). Attempts to obtain full-length sequences for V␤6.2 and
21 were unsuccessful. Start codons for translation are underlined.
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PIG V␤
The Journal of Immunology
2593
FIGURE 2. Amino acid sequence alignment of the porcine V␤ segments. Sequences are presented in a phylogenetic order. Gaps are indicated by
hyphens. Numbering starts at first residue of the mature protein. Important conserved residues are highlighted with an asterisk.
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FIGURE 3. Nucleotide sequences of the pig CDR3 region and J␤ segments. Numbering of the porcine CDR3 clones is indicated on the left of each
sequence. Putative V␤ 3⬘ terminus (C-term) and D segments bracketed by N regions (N-D-N) are grouped according to the J␤ segment usage.
2594
TCR ␤ DIVERSITY IN A PRECLINICAL MODEL OF ORGAN TRANSPLANTATION
from a class II-matched graft, accepted after a 12-day course of
cyclosporine, was tested for the distribution of V␤100 CDR3
length polymorphism (spectratyping). Fig. 6 illustrates the results
obtained from a pilot study performed on both PBL and renal
biopsies collected 30 days post transplantation from animal 11574.
The distribution of V␤100 CDR3 lengths in graft-infiltrating cells
showed the prevalence of some rearranged VDJ cDNA in this
animal. The clonal dominance among the V␤100 subfamily was
also observed in graft-infiltrating cells from another tolerant animal (Fig. 7). In addition, several V␤ spectratypes from intragraft T
cells for some other V␤ subfamilies presented a CDR3 length distribution without sign of clonal dominance (Fig. 6). Comparatively, the Gaussian distribution of the V␤100 CDR3 lengths in
PBL collected at the same time (Fig. 6) suggested that no selection/
dominance occurred in resting T cells.
Discussion
T cell V␤ gene repertoires have been extensively characterized in
rodents (17) and humans (16). Fragmentary information has also
been gathered on the V␤ sequences of cows (18), rabbits (19),
horses (20), and primates (21). Together with the human and rodent V␤ sequences, this set of porcine sequences represents one of
the largest collections of V␤ segments described so far. cDNA
sequences corresponding to 17 different functional V␤ subfamilies
and 12 distinct J␤ segments were established and showed closer
homology to their human counterparts. This number of functional
pig V␤ subfamilies is compatible with the notion that the diversity
of the expressed pig V␤ repertoire is of the same order of magnitude as the 25 human functional V␤ or the 20 V␤ defined in rodents (22). Sixteen of the 17 porcine V␤ subfamilies shared at least
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FIGURE 4. Alignments of pig J␤ nucleotide sequences with their human germline homologs. Hyphens indicate sequence identity between pig and
human. The human J␤ nomenclature is from GenBank.
The Journal of Immunology
FIGURE 5. Alignment of the pig germline D␤ region with the human
germline D␤2 and D␤1 regions. Hyphens indicate identity to the top sequence and dots correspond to gaps introduced to maximize the alignment.
The D region is boxed, the human D␤2 sequence is in bold characters, and
heptamer and nonamer signals are underlined.
FIGURE 6. V␤ CDR3 length spectratyping of
intragraft and peripheral lymphocytes. Total RNA
was isolated from PBL as well as from a wedge
kidney biopsy of a tolerant animal 11574, 29 days
post transplantation. Samples were then processed
as indicated in Materials and Methods. The y-axis
represents the intensity of the radioactive signal,
whereas the number of possible CDR3 lengths are
computed on the x-axis.
FIGURE 7. V␤100 CDR3 length spectratyping of intragraft T cells in
animals 11574 (top) and 11560 (bottom) 29 days post transplantation. Both
animals were tolerant to a MHC class I disparate renal allograft.
and V␤100 (Fig. 2) were identical with that of their murine counterparts (14, 23), suggesting that the differences observed in these
pig V␤ C-termini are likely to be germline encoded rather than
generated from somatic recombination events. The CGA sequence
within the C-terminal motif of the pig V␤2 (CGAM in Fig. 2) is
also possibly germline encoded, because it has been observed in
three independent pig clones 9 and 31 (Fig. 3), which all used the
same V␤2. The same C terminus has also been found in the mouse
(24), cattle (18), and horse (20) V␤2 homologs. The determination
of the predicted COOH terminus of the porcine V␤4, V␤7, and
V␤8 segments (Fig. 2) remains elusive due to lack of information
on the corresponding genomic sequences.
This study has identified 12 distinct pig J␤ segments (Figs. 3 and
4), a number close to the 13 human and 12 murine J␤ segments,
which are known to be organized into two separate clusters, each
associated with a unique set of D␤ (reviewed in Ref. 11). The
presence of pig J␤ sequences closely related to the human J␤1 and
J␤2 (Fig. 4) suggests a possible distribution of the pig J␤ in two
clusters. In addition, we found that three CDR3 regions containing
the J␤1.1, 1.2, or 1.3 had the upstream motif GGGACAGGG,
which is identical with the D␤1 sequence described in trout (25),
mouse (26), and human (27) (results not shown). This latter result
supports the existence of a D␤1 segment within the pig TCR␤
cluster. The cloning of a porcine germline D␤2 sequence (Fig. 5),
together with the identification of the D␤1 motif in several clones,
strongly argues the presence of two D␤ segments in miniature
swine. The duplication of the D␤ locus would imply that the pig J
segments are also organized in two clusters. This hypothesis is
supported by the presence of pig J␤ sequences similar to human
J␤1 segments in all pig functional TCR␤ clones containing a D␤1
segment (clones 2, 10, 14, and 16 in Fig. 3). The same restricted
association was seen for the pig D␤2 and J␤2 (data not shown).
If we assume that the overall organization of the pig TCR␤
locus is similar to that of human and mouse, we should also expect
to detect a second C␤ sequence in the pig TCR␤ locus. So far, only
one pig C␤ sequence has been reported (7), and our current data do
not allow us to conclude on this matter.
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62% nucleotide sequence homology with an already described human V␤ sequence (Table II), suggesting a common V␤ ancestor
gene to each family. In contrast, the pig V␤100 sequence showed
⬍30% homology with any human germline V␤ sequence, while
sharing 66% homology with the mouse V␤2 sequence. Furthermore, the pig V␤100 element appeared functional, because five
independent V␤ cDNA clones had this sequence rearranged in
frame with a J␤ segment (data not shown). Similar to the pig
V␤100, the murine V␤2 has only 40% homology to any human V␤
and has been described as the only rodent V␤ family with marked
divergence from other mammalian V␤ sequences (22). This divergence might be the result of an early drift of an ancestral V␤ gene
from the pool of otherwise closely related V␤ genes. However, the
fact that the V␤2 equivalent is found in both the pig and mouse,
but not in human, is in disagreement with this hypothesis. We
rather favor the view that the human V␤2 ancestor gene may have
been selectively deleted, possibly due to TCR autoreactivity.
The probability that PCR-generated errors may account for
some of the V␤ sequence variability is low because each V␤ sequence was reproducibly found in several clones in association
with different D-J segments. The fact that the 5⬘ ends of the whole
V␤ sequences were obtained independently from the 3⬘ V␤ portions for nine V␤ families also raises a legitimate concern of creating “mixed” sequences, resulting from the juxtaposition of 5⬘ and
3⬘ V␤ fragments originated from close members of the same V␤
subfamily. However, it should be noted that eight of the nine reconstructed pig V␤ sequences were derived from 5⬘ and 3⬘ sequences with identical overlaps of ⬎70 nucleotides in a region
where none of the human V␤ sequence homologs were identical.
Furthermore, very stringent PCR conditions were adopted to selectively extend the amplified strand from the oligonucleotide
primers, thereby limiting possible cross-hybridization to other
members of the same V␤ family.
Although the determination of the 3⬘ boundary of most pig V␤
segments was facilitated by the presence of the consensus CASS
sequence, usually found at the COOH termini of V␤ (16) (Fig. 2),
the COOH end of six pig V␤ sequences, V␤2, V␤4, V␤7, V␤8,
V␤20, and V␤100, markedly diverged from that of the consensus
sequence (Fig. 2). However, the C-terminal motifs of the pig V␤20
2595
2596
TCR ␤ DIVERSITY IN A PRECLINICAL MODEL OF ORGAN TRANSPLANTATION
Acknowledgments
We thank Drs. Gerry Waneck and John Iacomini for their critical review of
the manuscript and helpful discussion. We are also grateful to Dr. E. Pfaff
from the Federal Research Center for Animal Virus Diseases in Tübingen,
Germany, for providing us with the sequence of a pig TCR ␤ cDNA (clone
B4) that contained a V␤ sequence identical with our V␤17.
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Initial data from V␤100 spectratyping studies revealed a marked
dominance among V␤100 CDR3 lengths within the pool of intragraft lymphocytes in two animals (Fig. 7). Additional studies on
naive PBL demonstrate a Gaussian distribution of CDR3 lengths
observed in three naive animals (results not shown). These findings
demonstrate that the V␤100 segment is actually a functional entity
of the porcine T cell repertoire. In addition, the absence of V␤100
CDR3 length dominance in PBL collected at the same time (Fig.
6) may imply that infiltrating T cell clones are either selected at the
time of entrance to the kidney graft or selectively expanded after
entering the graft. Although further studies will be necessary to
definitely establish clonal dominance in this model, it is tempting
to suggest that this clonal selection could be related to the clinical
status of the graft.
In summary, the V␤, J␤, and D␤ sequences described in this
study represent a unique collection of molecular information that
demonstrates that the porcine TCR␤ locus is most likely organized
in a similar manner to that reported for rodents (11) and humans
(16). Given that the size of the porcine V␤ and J␤ gene pool may
be similar to that of other mammals, the description of 17 V␤
subfamilies and 12 J␤ sequences should be adequate to account for
most T cell ␤-chain diversity. A V␤100 segment, unique to swine
and rodents, was also described and appeared to be clonally dominant in cells infiltrating kidney graft. This study sets the groundwork for analysis of intragraft TCR specificities and correlations
with the clinical condition of the host in a preclinical model of
organ transplantation.