The Journal of Immunology, 2001, 166: 2589-2596.
Copyright © 2001 by The American Association of Immunologists
A Particular TCR 
Variable Region Used by T Cells Infiltrating Kidney Transplants1
Christophe Baron,
David H. Sachs and
Christian LeGuern2
Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129
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Abstract
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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 CD8+ 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 J1 and J2.
The fact that the pig J1 segments were always found joined to the
D1-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 V100, which exhibited similarity to
the murine V2 sequence but had no described V homolog in humans.
Pilot spectratyping studies on V usage revealed a clonal dominance
of V100+ T cell subsets among infiltrating cells in two
accepted grafts.
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Introduction
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Miniature
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 complementarity-determining 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 V100 segment.
Twelve J along with two D sequences were also described. This set
of porcine sequences, 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 V100+
cells in the intragraft subset of lymphocytes.
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Materials and Methods
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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 µg 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 V2 and V14 were
designed to anneal to sequence stretches conserved among human, rat,
cattle, and mouse V2 or V14 (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 c-DNA 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 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.
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 µg of total RNA was amplified in 25 µl final volume
containing 0.2 mM of each dNTP, 1.5 mM MgCl2, 50
mM KCl, 10 mM Tris-HCl (pH 9), 0.8 µM 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).
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Results
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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: V1, 6.1, 6.2,
7, 8, 12, 20, 21, 22.1, and 100. The use of V-specific degenerate
primers for the human V2 and V14 (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 V4,
V5, V10, V11, V17, V22.2, and V24 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, V6 and V22, 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 V100, 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).
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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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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
three-dimensional 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 35Tyr,
37Gln, and 92Ala, were
conserved in 85% of cases. The 90Tyr was
replaced by a Leu in the pig V100, similar to what is found in the
mouse V2 counterpart (14). Most of the porcine V
sequences segregated into the two V subgroups defined in other
species (8). Thus, the pig V1, 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
64Arg (8). Similarly, the V12,
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 V11 and V20 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
Arg in the CASR sequence of pig V12 (Fig. 2) is likely to be the
result of the V-J joining process, because the sequence of another
independently cloned V12 cDNA (clone 6, Fig. 3) also predicted a CASS sequence in that
region.
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 J1.5 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 111Leu
or Val or Tyr in position 111, which are essential to the V
/V and
V/C interactions, respectively. The motif TTC/T-GGN-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 homology 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 D2 corresponding region, and only 50% to the human D1
region, indicating that this porcine D region was likely the homolog of
the human genomic D2 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 V100+ T lymphocytes within
the intragraft cell pool
Because the V100 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 from a class
II-matched graft, accepted after a 12-day course of cyclosporine, was
tested for the distribution of V100 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 V100 CDR3 lengths in graft-infiltrating cells showed
the prevalence of some rearranged VDJ cDNA in this animal. The clonal
dominance among the V100 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 V100 CDR3
lengths in PBL collected at the same time (Fig. 6) suggested that no
selection/dominance occurred in resting T cells.
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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.
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FIGURE 7. V100 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.
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Discussion
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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 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 V100 sequence showed <30% homology with any
human germline V sequence, while sharing 66% homology with the
mouse V2 sequence. Furthermore, the pig V100 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 V100, the murine V2 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 V2 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 V2 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, V2, V4, V7, V8, V20, and
V100, markedly diverged from that of the consensus sequence (Fig. 2). However, the C-terminal motifs of the pig V20 and V100 (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 V2 (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 V2. The same C terminus has
also been found in the mouse (24), cattle
(18), and horse (20) V2 homologs. The
determination of the predicted COOH terminus of the porcine V4,
V7, and V8 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 J1 and
J2 (Fig. 4) suggests a possible distribution of the pig J in two
clusters. In addition, we found that three CDR3 regions containing the
J1.1, 1.2, or 1.3 had the upstream motif GGGACAGGG, which is
identical with the D1 sequence described in trout (25),
mouse (26), and human (27) (results not
shown). This latter result supports the existence of a D1 segment
within the pig TCR cluster. The cloning of a porcine germline D2
sequence (Fig. 5), together with the identification of the D1 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 J1
segments in all pig functional TCR clones containing a D1 segment
(clones 2, 10, 14, and 16 in Fig. 3). The same restricted association
was seen for the pig D2 and J2 (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.
Initial data from V100 spectratyping studies revealed a marked
dominance among V100 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
V100 segment is actually a functional entity of the porcine T cell
repertoire. In addition, the absence of V100 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 V100 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.
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Acknowledgments
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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 V17.
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Footnotes
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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.
3 Abbreviation used in this paper: CDR3, complementarity-determining region 3.
Received for publication July 28, 2000.
Accepted for publication November 21, 2000.
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Abstract
Introduction
