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, in Rainbow Trout1 ,2
Basel Institute for Immunology, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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gene from an ectothermic source
using a degenerate primer for Ig superfamily V domains. Similar to
homologues in higher vertebrates, the rainbow trout CD8 gene encodes
a 204-aa mature protein composed of two extracellular domains including
an Ig superfamily V domain and hinge region. Differing from mammalian
CD8 V domains, lower vertebrate (trout and chicken) sequences do not
contain the extra cysteine residue (C strand) involved in the abnormal
intrachain disulfide bridging within the CD8 V domain of mice and
rats. The trout membrane proximal hinge region contains the two
essential cysteine residues involved in CD8 dimerization ( or
ß) and threonine, serine, and proline residues which may be
involved in multiple O-linked glycosylation events.
Although the transmembrane region is well conserved in all CD8
sequences analyzed to date, the putative trout cytoplasmic region
differs and, in fact, lacks the consensus p56lck motif
common to other CD8 sequences. We then determined that the trout
CD8 genomic structure is similar to that of humans (six exons) but
differs from that of mice (five exons). Additionally, Northern blotting
and RT-PCR demonstrate that trout CD8 is expressed at high levels
within the thymus and at weaker levels in the spleen, kidney,
intestine, and peripheral blood leukocytes. Finally, we show that trout
CD8 can be expressed on the surface of cells via transfection.
Together, our results demonstrate that the basic structure and
expression of CD8 has been maintained for more than 400 million
years of evolution. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T
cell receptors (1). This recognition process also involves
the TCR coreceptor molecules, CD8 and CD4, which bind to class I and II
molecules, respectively. Expression of CD8 and CD4 is critical for
thymocyte education and cell-mediated immune surveillance
(2). CD8 is a membrane-bound glycoprotein found on
cytotoxic T cells consisting of either CD8 homodimers or
CD8ß heterodimers. Both chains ( and ß) are composed of a
single extracellular Ig superfamily
(IgSf)4 V domain, a
membrane proximal hinge region, a transmembrane domain, and a
cytoplasmic tail. An essential role for CD8 during thymocyte
development was demonstrated by gene targeting, as selection of
competent peripheral cytotoxic T cells was greatly reduced in
CD8-/- mice (3). Moreover,
CD8 expression is absolutely required for expression of the
ß-chain (4).
The ability of CD8 to act as a TCR coreceptor lies in its capacity to
interact with MHC class I and ß2-microglobulin
(ß2m) during TCR-mediated MHC peptide
recognition (5, 6, 7, 8). Indeed, CD8 associates with
ß2m and the 2 and 3 domains of MHC class
Ia molecules using its A/B ß strands and the
complementary-determining regions (CDR) within the extracellular IgSf V
domain. This association increases the adhesion/avidity of the T cell
receptor with its class I target. Thus, CD8 is an active participant in
the T cell recognition process. In addition, CD8 associates with the
src tyrosine protein kinase p56lck through a
conserved binding motif within the cytoplasmic tail of CD8 (9, 10). Not only does CD8 stabilize TCR/MHC class I contact, the
interaction of CD8 and TCR with MHC class
I/peptide/ß2m results in the phosphorylation of
the TCR by p56lck. This latter event leads to the
rapid activation of the cytotoxic T lymphocyte via internal signaling
events. A similar lck-binding motif is found within the cytoplasmic
tail of CD4, but in contrast CD8ß does not associate with
p56lck due to the absence of this motif.
The CD8 and ß genes are tightly linked (36 kbp apart in mice
(11)) within the same overall linkage group as Ig
in
mice and humans (12, 13, 14), suggesting that the CD8 locus
might have arisen via a cis-duplication event involving the
Ig locus. In humans and mice, alternative splicing gives rise to
CD8 variants which either lack the transmembrane domain (humans) or
a portion of the cytoplasmic region (mice) (15, 16).
Secreted forms of CD8 have been identified in humans although the
role of secreted CD8 is not known. In mammals, CD8 is expressed on
the majority of thymocytes, 
30% of peripheral T lymphocytes (mainly
ß heterodimers), intraepithelial lymphocytes (mainly
homodimers) and on some NK and dendritic cell populations
(2). CD8ß heterodimers are solely expressed on
TCRß+ T cells in most mammals although
chicken intraepithelial lymphocyte CD8ß subsets are largely
TCR
+ (17).
Cell-mediated responses (18) and molecules associated with
the cellular immune response have been studied in trout including the
description of TCRß, MHC class Ia and Ib, TAP, LMP, and MHC class
IIß sequences (19, 20, 21, 22, 23). We now describe the cloning and
characterization of CD8-encoding sequences from rainbow trout and
present the overall structural composition, genomic organization, and
tissue-specific expressions of trout CD8. Surprisingly, a motif in
CD8 previously thought to be critical for proper CTL maturation and
selection is lacking in this cold-blooded vertebrate.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rainbow trout, Oncorhynchus mykiss (ARO-F2, Idaho origin), were obtained from Aquatic Research Organisms (Hampton, NH) and maintained in 14°C water at the Basel Institute for Immunology. Killing was accomplished using 100 µg/ml MS-222 (Norvartis Pharmaceuticals, Basel, Switzerland) supplemented with 150 µg/ml sodium bicarbonate. Isogenic trout (OSU clonal line 1-14 and HC clonal line E1B) have been described elsewhere (24).
cDNA cloning and genomic organization
A degenerate primer corresponding to highly conserved residues
(D-E/S-G-X-Y-F/Y/I-C) within the F strand of IgSf V domains was
synthesized. This reverse primer (5'-CARWWRTAIIINCCNIHRTC-3', where
R = a/g, W = a/t, Y = c/t, H = a/c/t, N =
a/c/g/t, and I = inosine) plus a T3 anchored (5' region of the
pCMV-ZAP Express MCS) primer were used to amplify V-like domains from a
trout thymocyte ZAP Express unidirectional cDNA library
(25). PCR amplification conditions were as follows: 94°C
for 15 s, 45°C for 30 s (+0.2°C/cycle), and 72°C for
30 s, with a final extension of 2 min at 72°C. Products were
cloned into pCRII (Invitrogen, San Diego, CA) and sequenced. Base pairs
refer to positions within Onmy-CD8 cDNA (AF178053). Two
sense primers (E1S, 5'-GAGCTTGAACGTGTTGCTGT-3'; bp 1–20) and
nested ES2, 5'-AGAGGGTGGAGATCACTTGT-3'; bp 126–145) based on a
putative V region cDNA were used in conjunction with an anchored T7
primer (3' region of the pCMV-Zap Express MCS) to amplify (30 cycles
consisting of 94°C for 10 s, 55°C for 30 s, and 72°C
for 1 min) the full-length cDNAs from the trout thymocyte cDNA library.
Products were cloned into pBlunt (Invitrogen) and sequenced.
Full-length cDNAs were also amplified from thymocyte first-strand cDNA
by RT-PCR using the E1S and the 3' untranslated terminal region (UTR)-R
primer. Trout CD8 genomic clones were amplified from 200 ng of trout
genomic DNA (gDNA) by PCR (Elongase; Life Technologies, Rockville, MD)
using the forward 5' UTR primer (E1S) and a reverse primer located
within the 3' UTR (3' UTR-R, 5'-ACTGCAGAGCTTTTGTCTTTG-3'). Long
range PCR conditions consisted of 2 min at 95°C followed by 30 cycles
of 94°C for 30 s, 58°C for 30 s, and 68°C for 4 min.
The amplified product was cloned into pBlunt, sequenced, and compared
with trout CD8 cDNAs to determine exon/intron boundaries. Nucleotide
differences were not found between OSU and HC clonal lines. Allotypic
differences found between cDNAs were confirmed by sequencing the
genomic DNA from the same ARO-F2 fish.
Southern and Northern blotting
Genomic DNA and RNA isolation and blotting protocols have been
described elsewhere (25, 26). For both Southern and
Northern blotting, a portion of the variable region (317 bp) of trout
CD8 was amplified (E2S, 5'- GAAACTCTCCAACTGAGTTCT-3'; bp 85–105,
and E2R, 5'-TCGAGTTACTTCACCAAACAC-3'; bp 382–402),
randomly labeled (BRL- Life Technologies, Gaithersburg, MD) with
[32P]dCTP (Amersham, Arlington Heights, IL) and
used as a probe under stringent conditions (0.25x SSC/0.25% SDS
68°C final wash). PCR conditions were identical to those for CD8
RT-PCR.
Transient transfection
COS-7 cells were maintained in IMEM supplemented with 5%
heat-inactivated FCS (Life Technologies) and kanamycin. The
extracellular, transmembrane, and cytoplasmic domains of
Onmy-CD8 were amplified (PFU; Stratagene, La Jolla, CA)
using a forward
(5'-GAGTCAAGCTTCAAGAAACTCTCCAACTGAGT-3', bp 82–103)
and reverse primer (5'-AGCTAGGTACCTTAGAAAAGTCTGTTGTTGGC-3',
bp 691–711) containing HindIII and KpnI
restriction sites, respectively (underlined). The amplified fragment
was purified (Qiaquick PCR spin column; Qiagen, Basel, Switzerland),
digested with HindIII/KpnI, and ligated into the
HindIII/KpnI site of pFlag-CMV1
(N-terminal Flag; Sigma, St. Louis, MO). COS-7 cells were mock
transfected or transfected with 3 µg of the pFlag-OmCD8
construct in 60-mm plates (70% confluency) using the Superfect
protocol (Qiagen). Two days posttransfection, cells were harvested,
washed twice in PBS containing 2% FCS, and adjusted to
107 cells/ml. Fifty microliters of cells was
stained with 5 µg/ml of the M2 anti-Flag mAb (Sigma) in PBS/FCS,
washed three times in PBS/FCS, incubated with goat anti-mouse
IgG1-FITC at 5 µg/ml (Southern Biotechnology Associates, Birmingham,
AL) in PBS/FCS, washed three times in PBS/FCS, and resuspended in
PBS/FCS containing 0.1% sodium azide. As an additional negative
control, cells were stained with only the secondary Ab. Propidim iodide
was added and the cells were then analyzed (live gate) for surface
expression using a FACSscan (Becton Dickinson, Mountain View, CA) flow
cytometer.
RT-PCR
First-strand cDNA template preparations have been described
previously (26). First-strand cDNAs were generated from
500 ng of total RNA in a 20-µl reaction. For RT-PCR analysis, the E1S
forward primer located in exon 1 was used in conjunction with an exon 2
reverse primer (E2R) to amplify (25 cycles) trout CD8 transcripts.
Amplification conditions consisted of 94°C for 15 s, 58°C for
30 s, 72°C for 30 s, and a final incubation at 72°C for 5
min using 1 µl of template (except for thymus 1:10). Products were
electrophoresed (2% agarose), blotted to Hybond
N+ (Amersham) under alkaline conditions (0.4 N
NaOH), and hybridized with an internal V region probe (as described in
Southern and Northern blotting). As a control of template quality,
EfTu-1 transcripts were amplified (29 cycles) using previously
described primers and conditions (26).
Sequencing and phylogenetic analysis
cDNA and gDNA clones were sequenced by dideoxy chain termination
chemistry using universal and gene-specific infrared primers (MWG
Biotec, Ebersberg, Germany) in conjunction with the Thermo Sequenase
kit (Amersham). Sequences were processed via an automated sequencer
(LI-COR 4000L). Putative signal peptides and transmembrane and
cytoplasmic regions were based on SMART predictions (27)
and on the crystal structure of human CD8 (28).
Alignments, phylogenies, and bootstrapping were conducted using the
Clustal X software package (29). Transcription factor
motifs and stem loops were predicted using Signal Scan 4.05
(30), MatInspector 2.2 (31), and Stem Loops
(http://www.molgen.uc.edu/analyze/).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNAs
A degenerate reverse primer corresponding to highly conserved
residues within the "F" strand of V-set IgSf members was used to
identify new V regions from a rainbow trout thymocyte unidirectional
cDNA library. A variety of products were cloned, sequenced, and
analyzed by BlastX searches using the entire GenBank database as well
as a teleostei (bony fish) subdatabase directory. One clone caught our
attention due to its weak but definite identity to V regions of
TCRß and , IgH, and Ig from both bony fish and mammalian
cDNA sources (25–28% identity).
Since this fragment probably corresponded to the 5' end of an authentic
V region-encoding gene, forward primers were synthesized and used to
amplify the 3' portion of this gene coupled with an anchored T7 vector
primer. A single product (1082 bp minus vector contributions) was
amplified, sequenced, and once again subjected to a BlastX search. The
clone was most similar to the complete chicken CD8 chain
(6e-12) followed closely by full-length CD8
cDNA sequences from mammalian species
(5e-11–3e-5). Following
the sources for CD8, the next BlastX/P scores were for V regions of
TCR (1e-4) and CD8ß
(2e-4).
Onmy-CD8 (AF178053) encodes a single open reading frame
of 226 aa followed by a short 3' untranslated region (370 bp)
including a polyadenylation site and tail. After cleavage of the
putative 22-aa hydrophobic leader sequence, a predicted mature protein
of 22 kDa (204 aa) would be generated, not including
posttranslational modifications. Thus, trout CD8 is smaller than
other vertebrate CD8 chains which average 217 aa for their mature
forms.
Structural analysis of CD8
An alignment was assembled (Fig. 1
)
using all available CD8 amino acid sequences and our new clone,
Onmy-CD8, to display conservation of residues and domains
found within CD8 from species ranging from fish to humans. Alignment
of the mature proteins reveals an average of 30% identity across
this diverse group of species.
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Cysteine residues (human C-22/C-94) involved in the canonical
disulfide bonding to form the V domain are absolutely conserved but, as
previously reported for chicken CD8, an extra internal cysteine
residue (C-33) responsible for the unique intradomain disulfide bond
found in mice and rats is not present in the chicken (17)
or trout sequences. This unusual C-22/C-33 bond was not found in the
crystal structure of human CD8, although C-33 is indeed present
within the human sequence (28).
CD8 associates with MHC class I-peptide-ß2m
complexes via the A/B ß strands and CDR regions found within the IgSf
V domain. The A strand is poorly conserved whereas the B strand
displays a higher level of similarity among the vertebrates.
Additionally, the putative CDR1 and 2 regions are highly variable,
whereas the CDR3 region essentially contains residues with either
nonpolar or uncharged polar side chains. Interestingly, while cloning
CD8 cDNAs from three strains of trout, we found an allotypic variant
(AF178054) containing 3 bp differences within the V domain resulting in
two coding substitutions (N55
D and
H58N, Fig. 1
). These two substitutions result
in a charge shift located in a loop region between the C'' and D
strands which form a portion of the CDR2 implicated in binding MHC
class I. Additionally, the diglycine bulge (GXG) found in the G strand
of CD8ß is lacking in all CD8 strands, including trout.
CD8 is capable of forming both homo- () and heterodimers
(ß, with CD8ß) based on disulfide bonding of conserved cysteines
within the extracellular hinge region. All species display conservation
for these two canonical residues (human C-143/160), suggesting that
trout CD8 is also capable of dimerization. In addition, this region
contains multiple O-linked glycosylation sites (XPXX,
glycosylated if X = S or T) (32, 33). Sialation of
the O-linked sugar residues along with the proline residues
are thought to keep the hinge in an extended configuration and to repel
it from the membrane surface, thus allowing CD8 to reach the 2
and 3 domains of MHC class I (28). This basic scheme
has been well preserved during evolution since the trout hinge region
like that of other vertebrates is also rich in serine, proline,
and threonine residues that constitute several O-linked
glycosylation sites (T110, T117,
T120, and possibly
T142). N-linked glycosylation sites
are not found in the trout CD8 sequence.
Transmembrane and cytoplasmic domains
Overall, the CD8 transmembrane domain (23 aa) retains the
highest level of identity (39%) among the various vertebrates,
including an absolute conservation of a WAPL (trout aa 150–153)
sequence motif. Perhaps more importantly, a conserved motif (CXCP)
within the cytoplasmic domain of CD8 is thought to be responsible
for binding p56lck (9). Chickens
offer a variant of this motif (CXCK) at the same location within the
alignment, but the motif is missing in the trout sequence. The first
cysteine of the sequence (human C-194) is conserved but an apparent
insertion distorts the remainder of the motif. Instead, a similar
motif, CXCN169–172, is found at the very
beginning of the predicted trout cytoplasmic domain which may serve as
an lck homologue docking site, although this is unlikely due to the
positioning of this motif next to the membrane border.
Phylogeny of CD8
Phylogenetic analysis (Fig. 2) was
conducted using CD8 and ß mature sequences and CD7, all of which
have the same basic structural composition. As depicted in the
neighbor-joining tree, trout CD8 relates best to the CD8 and
ß-chain groups, preferentially clustering between the chicken CD8
and ß-chains as supported by bootstrap analysis.
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Primers located within the 5' and 3' untranslated regions were
used to amplify a genomic fragment (2.3 kb, AF178055) containing the
trout CD8 locus (Fig. 3). Similar to
other IgSf member encoding genes, all of the trout CD8 introns split
codons between the first and second nucleotide (type 1) with the
exception of the fifth intron which splits the codon from exons 5 and 6
between the second and third nucleotide (type 2). Exon 1 encodes the 5'
UTR and the majority of the predicted hydrophobic leader sequence, exon
2 the IgSf V-like domain, exon 3 the majority of the membrane proximal
hinge region, exon 4 the hydrophobic transmembrane region, and exons 5
and 6 together code for the cytoplasmic domain. Overall, the trout
CD8 genomic organization is nearly identical to that of the human
and mouse, except that in the mouse, the leader and V domain form a
single exon (Fig. 3B) (15, 16).
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gene, one located in intron 4
and two within intron 5. A putative T cell-specific enhancer was later
found in the last intron of the CD8 gene, located near a large stem
loop structure (34, 35). When introduced into transgenic
mice, this enhancer region was able to drive expression in an NK
cell-specific manner, which may be related to the usage of CD8
homodimers for this cell type (36). Within the trout
locus, several regulatory motifs implicated in cellular immunity were
found within the introns including binding sites for c-ets, GATA-3, and
Ikaros (Fig. 3A). Additionally, introns 1–4 each contain
sequences capable of forming stem loop structures. Interestingly,
intron 4 contains six copies of an imperfect 30-bp repeat, which is
similar to both RNA polymerase II and an adhesive protein found in
Mytilus (accession number S68957) by BlastX inspection. In addition,
this region (intron 4) contains a variety of transcription motifs
(CCAAT box, AP-1, NF-Y, and CDPC binding sites) which are typically
found in promoter elements, followed by a TATA box at position
1665. Southern blot analysis
Based on the domain boundaries of trout CD8, we amplified the
IgSf V domain (exon 2) and used it as a probe for Southern blotting
(Fig. 4). One to three bands can be
observed for each individual and digest, suggesting that the trout
CD8 gene exists as a single copy and not as a member of a multigene
family. In addition, EcoRV and HindIII digests
indicate the presence of polymorphic variants for trout CD8.
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The expression pattern of trout CD8 was first analyzed by
Northern blotting (Fig. 5A).
An intense thymic signal was found at a size correlating with the cDNA
length (1 kb message). Two other bands were observed within the
thymus which likely represent nonprocessed heterogeneous nuclear RNAs
for CD8 as has been observed in mammals. For a more sensitive
examination of CD8 expression, we utilized RT-PCR (Fig. 5B). As shown by Northern blotting and RT-PCR, the thymus is
the major source of CD8 expression, followed by the spleen,
intestine, kidney, and peripheral blood leukocytes. Weak signals were
also detected in the testis and heart by RT-PCR, probably due to a few
circulating CD8+ cells in these tissues.
Additionally, by using a set of primers located in exons 3
and 5, we were unable to detect any splice variants similar to those
found in humans which result in the deletion of the transmembrane
domain (data not shown). Finally, we examined whether
Onmy-CD8 can be expressed on the surface of
transiently transfected cells (Fig. 5C). A
moderate level of surface expression (27% positive) was
observed on cells transfected with the pFlagOmCD8
construct as detected with the M2 anti-Flag mAb confirming the type
1 nature of this protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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homologue from this species. Comparison of trout CD8
with other CD8 sequences indicates that the basic structure of this
molecule has been preserved during more than 400 million years of
evolution. One notable divergence is that trout CD8 lacks the
previously described consensus cytoplasmic motif that is critical for
association with the protein tyrosine kinase
p56lck. Our data also establish that the basic
genomic organization has been fairly rigorously maintained for CD8.
Also, as would be expected, the major source of CD8 expression is
the thymus.
Several groups have demonstrated that the IgSf variable region of
CD8 mediates binding of CD8 with the 2 and 3 domains of MHC
class I molecules (5, 7, 8). Three key positions have been
identified in the HLA 2 domain (Q-115, D-122, and E-128) that are
critical for the interaction of MHC class I with the A and B strands
found within the V domain of CD8, presumably via electrostatic
mechanisms (7). In a recent study (21), we
characterized three major lineages of MHC class Ia alleles in trout
which maintain identity with HLA Q-115 and D-122, suggesting that these
sites could also be involved in CD8-MHC recognition in fish. Although
either leucine or lysine is found at the trout position corresponding
to human glutamic acid (128>), trout class I molecules do
have an aspartic acid shifted by just one position from this site.
These trout residues (QDD) are found in the ß2 and ß3 strands of
the 2 domain, as are the Q-115, D-122, and E-128 residues of HLA.
Arginine (4), lysine (21), and leucine
(25) found within the A and B strands of human CD8
(Fig. 1) make contact with ß2m and the
conserved residues in the 2 domain of HLA-A2. These residues
(excluding lysine (21) are well conserved among the
various species, except that rat and trout lack the arginine at the
beginning of the A strand. Leucine (25) of human CD8
makes contact with lysine (58) found within the DE loop of
ß2m based on side chain interactions. In
chicken and trout CD8, this position is encoded by phenylalanine or
valine, both of which possess nonpolar side chains and differing from
most ß2m sequences, the conserved lysine
(58) position is replaced by glutamine in both chickens
and trout. It should be noted that all species, including trout,
contain basic amino acids in the A and B strands, and thus positively
charged residues within these strands are probably required for the
association of CD8 with invariant residues in the MHC class I 2
domain.
An exposed acidic loop
(D223–E229) within the
3 domain of MHC class I makes contact with the CDR loops found in
CD8 (5, 7, 8). When not bound by CD8 or
CD8ß, the 3 acidic loop is very flexible but it takes on a more
rigid structure when bound by CD8. Human CD8 lysine
(58) within the CDR2 loop is critical for the
electrostatic-based association with the 3 acidic loop of HLA.
Mutational analysis of the human CD8 CDRs resulted in >70–80%
(CDR1) and 50–65% (CDR2) reduction in MHC class I binding
(6). Moreover, introduction of negatively charged residues
within CDR1 abrogated HLA binding, presumably via electrostatic
repulsion with the HLA 3 acidic loop. The trout CDR1 and 2 regions
each contain basic residues that could interact with the negatively
charged 3 loop found in trout class I molecules (Fig. 6).
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allotypes possess two amino acid
replacements located at the distal end of the putative CDR2 region
which result in a charge shift. In mice, two allotypic variants for
CD8 have been identified which differ by a single amino acid
substitution in the V-domain which in fact constitutes the basis for
serological markers that are capable of distinguishing the two
allotypes (15). In chickens, there are three allelic
variants for CD8, and the majority of amino acid replacements are
found within the CDRs. The replacement to synonymous ratios within this
study suggested a strong selective pressure for the CDRs which may be
related to pathogen avoidance (37). Alternatively, the
trout CD8 allotypes could be involved in recognizing the two
different trout MHC class I 3 acidic loops which differ in net
charge (Fig. 6) and would suggest that the trout CD8 has coevolved with
the class I molecules. We are currently addressing this issue via
segregation analyses. Finally, mutational analysis and crystal data
have also implicated human asparagine (99) in the CDR3
loop as making contact with a specific residue
(E229) in the acidic loop of HLA (6, 8). In trout CD8, two asparagine residues (N93,
95) are found in this area and the trout MHC class I 3
loop variants each contain the conserved glutamic acid residue,
implying that the trout CDR3 loop of CD8 may recognize the acidic
loop via similar means. The wide variability found within both the A/B
strands and CDRs (Fig. 1) among the various vertebrate classes probably
dictates species-specific interaction(s) of CD8 and MHC class
I.
By far the most surprising finding in this study was that the trout
CD8 chain lacks the consensus p56lck tyrosine
kinase motif critical for CD8 association. The alliance of
p56lck with CD8 leads to the subsequent
phosphorylation of the TCR by lck in higher vertebrates upon class I
interaction. Although the chicken CD8 lck motif deviates slightly
from the consensus sequence, coimmunoprecipitation kinase assays led to
the identification of a chicken lck homologue that associates with the
cytoplasmic tail of chicken CD8 (38). Interestingly,
neither trout nor chicken possess serine residues thought to be
critical for lck dissociation. Transgenic mice expressing a tailless
form of CD8 (CD8-/-
background) have a more severe phenotype than do mice carrying a
transgene in which the CXCP motif was altered by mutagenesis. Overall,
CD8 tailless mice have three to four times fewer
CD8+ peripheral T cells, and their ability to
mount an efficient antiviral CTL-mediated response is reduced in
comparison to the CXCP mutated mice, suggesting that other factors with
signaling capabilities might associate with the cytoplasmic domain
(4, 39, 40). Recently, Trede and Zon (41)
described in situ staining patterns within the zebra fish thymus using
an lck probe, thus an lck homologue is present in teleost fish.
Although the trout CD8 cytoplasmic tail lacks the full consensus lck
motif, it may still associate with an lck homologue based on the highly
charged proximal portion (R/K-X-R-X-R/K-C) of the consensus motif.
In all vertebrate species, the thymus appears to be the major site of T
cell lymphopoiesis and education (42). This is supported
in part by the fact that the teleost thymus expresses high levels of
RAG-1 and RAG-2, TdT, Ikaros, TCR/ß, class Ia and II, lck, and now
CD8. At the current time, well-defined mAbs to trout T cells are
nonexistent. Thus, the cloning of trout CD8 provides a new tool for
addressing the involvement of CTLs during an immune response in fish,
and also opens an avenue for examining positive and negative selection
in an ectothermic model. Obviously, one aspect to be pursued is whether
the cytoplasmic region of trout CD8 associates with unique signaling
components (protein tyrosine kinases or protein kinase C) other than
p56lck for proper T cell development and
activation.
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Acknowledgments |
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Footnotes |
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2 The sequences described in this report have been deposited in GenBank under the following accession numbers: AF178053-8055.
3 Address correspondence and reprint requests to Dr. John D. Hansen, Basel Institute for Immunology, 487 Grenzacherstrasse, CH-4005 Basel, Switzerland. E-mail address:
4 Abbreviations used in this paper: IgSf, Ig superfamily; ß2m, ß2-microglobulin; CDR, complementary-determining region; UTR, untranslated terminal region; DNA, genomic DNA.
Received for publication November 11, 1999. Accepted for publication December 30, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 and 3 domains of MHC class I. J. Exp. Med. 182:1275. and HLA-A2. Nature 387:630.[Medline]
locus consists of two copies that are organized in opposite polarity. Genomics 16:503.[Medline]
locus is located on the telomere side of the immunoglobulin locus at a distance of 2 Mb. Genomics 16:512.[Medline]
: characterization of the human CD8 gene. J. Immunol. 142:3312.[Abstract]
ß and CD8 isoforms in chickens reveals a major TCR- CD8ß subset of intestinal intraepithelial lymphocytes. J. Immunol. 154:4485.[Abstract]
-chain in a teleost fish. J. Immunol. 157:207.[Abstract]
chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains. Int. Immunol. 4:215. gene. Mol. Cell. Biol. 13:7056. expression in NK cells but not cytotoxic T cells of transgenic mice. Int. Immunol. 8:1617., but not CD8-ß. Immunogenetics 46:396.[Medline]
-Lck association. Science 261:1581.This article has been cited by other articles:
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, possible
O-linked glycosylation sites for the trout. Plus signs
(basic charge) and filled ovals mark the residues comprising the
p56lck motif within the cytoplasmic region. Accession
numbers: chicken (I50610), rat (07725), mouse (P01731), bovine
(P31783), feline (P41688), canine (P33706), Orangutan (X60223, note
hinge deletion), and human (M12828). Numerical designations refer to
human CD8
