Description of an Ectothermic TCR
Coreceptor, CD8 α, in Rainbow Trout
John D. Hansen and Pamela Strassburger
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Copyright © 2000 by The American Association of
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References
J Immunol 2000; 164:3132-3139; ;
doi: 10.4049/jimmunol.164.6.3132
http://www.jimmunol.org/content/164/6/3132
Description of an Ectothermic TCR Coreceptor, CD8␣, in
Rainbow Trout1,2
John D. Hansen3 and Pamela Strassburger
C
ytotoxic and helper T cells recognize endogenously and
exogenously derived peptides presented by MHC class I
and II molecules via their ␣␤ 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 membranebound glycoprotein found on cytotoxic T cells consisting of either
CD8␣␣ homodimers or CD8␣␤ heterodimers. Both chains (␣ and
␤) 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 ␤-chain (4).
The ability of CD8 to act as a TCR coreceptor lies in its capacity
to interact with MHC class I and ␤2-microglobulin (␤2m) during
TCR-mediated MHC peptide recognition (5– 8). Indeed, CD8␣ associates with ␤2m and the ␣2 and ␣3 domains of MHC class Ia
molecules using its A/B ␤ strands and the complementary-deter-
Basel Institute for Immunology, Basel, Switzerland
Received for publication November 11, 1999. Accepted for publication December
30, 1999.
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
The Basel Institute for Immunology was founded and is totally supported by Hoffmann-LaRoche AG, Basel, Switzerland.
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: [email protected]
Abbreviations used in this paper: IgSf, Ig superfamily; ␤2m, ␤2-microglobulin;
CDR, complementary-determining region; UTR, untranslated terminal region; DNA,
genomic DNA.
4
Copyright © 2000 by The American Association of Immunologists
mining 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/␤2m 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 CD8␤ does not associate with p56lck due to
the absence of this motif.
The CD8␣ and ␤ genes are tightly linked (36 kbp apart in mice
(11)) within the same overall linkage group as Ig␬ in mice and
humans (12–14), 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 ␣␤ heterodimers), intraepithelial lymphocytes (mainly ␣␣ homodimers)
and on some NK and dendritic cell populations (2). CD8␣␤ heterodimers are solely expressed on TCR␣␤⫹ T cells in most mammals although chicken intraepithelial lymphocyte CD8␣␤ 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 TCR␣␤, MHC class Ia and Ib, TAP, LMP, and
MHC class II␤ sequences (19 –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
0022-1767/00/$02.00
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We have cloned the first CD8␣ 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 ␣␤) 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. The Journal of Immunology, 2000, 164: 3132–3139.
The Journal of Immunology
CTL maturation and selection is lacking in this cold-blooded
vertebrate.
Materials and Methods
3133
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.
Animals
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 ␮g/ml MS-222 (Norvartis Pharmaceuticals, Basel, Switzerland) supplemented with 150 ␮g/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
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⬘-TCG
AGTTACTTCACCAAACAC-3⬘; bp 382– 402), randomly labeled (BRLLife Technologies, Gaithersburg, MD) with [32P]dCTP (Amersham, Arlington Heights, IL) and used as a probe under stringent conditions (0.25⫻
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⬘-GAGTCAAGCT
TCAAGAAACTCTCCAACTGAGT-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 ␮g 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 ␮g/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 ␮g/ml (Southern Biotechnology Associates, Birmingham, AL) in PBS/FCS, washed three times in
First-strand cDNA template preparations have been described previously
(26). First-strand cDNAs were generated from 500 ng of total RNA in a
20-␮l 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 ␮l 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 (LICOR 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/).
Results
Isolation of trout CD8␣ 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 TCR␣␤ 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 fulllength 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 CD8␤ (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
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A degenerate primer corresponding to highly conserved residues (D-E/SG-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 OnmyCD8␣ cDNA (AF178053). Two sense primers (E1S, 5⬘-GAGCTT
GAACGTGTTGCTGT-3⬘; bp 1–20) and nested ES2, 5⬘-AGAGGGTG
GAGATCACTTGT-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⬘-ACTGCA
GAGCTTTTGTCTTTG-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.
RT-PCR
3134
CD8␣ IN TROUT
proteins reveals an average of ⬃30% identity across this diverse
group of species.
The extracellular variable and hinge domains
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-␤2m complexes via
the A/B ␤ 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 (N553 D and H583 N, 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 CD8␤ is lacking in
all CD8␣ strands, including trout.
CD8␣ is capable of forming both homo- (␣␣) and heterodimers
(␣␤, with CD8␤) 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
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FIGURE 1. Clustal X alignment of amino acids from CD8␣ chain precursors. Regions corresponding to the putative leader, IgSf V domain, hinge,
transmembrane, and cytoplasmic regions are shown in bold above the sequences. Dots indicate identity to the human sequence and dashes are used to
maximize the alignment. Region borders are denoted with solid black vertical bars. ␤ strands A–G, comprising the Ig fold within the V domain, are indicated
by underlining the human sequence. The CDRs (CDR1–3 based on Refs. 6 and 8) involved in MHC class I contact are designated by overlining the human
sequence. f, 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␣ for which the crystal structure is known
(28) and trout (underlined). TM, transmembrane.
The Journal of Immunology
3135
FIGURE 2. Phylogenetic tree of CD8
and CD7. An amino acid alignment
(Clustal X) of the mature proteins was
used to generate the unrooted Neighborjoining tree. Node values represent bootstrap analysis of 2000 replicants. CD8␣
accession numbers are found in Fig. 1. Other
accession numbers are as follows: CD7, human (X06180); mouse (D10329); CD8␤, feline (AB000484); gorilla (P30434); human
(Y00805); mouse (M22070); rat (P05541);
and chicken (Y11474).
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 ␤
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 ␤-chain groups, preferentially
clustering between the chicken CD8␣ and ␤-chains as supported
by bootstrap analysis.
Genomic analysis
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).
At the human CD8 locus, DNase I hypersensitivity studies revealed three hypersensitive sites within the CD8␣ 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␣.
RNA and surface expression
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
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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.
CD8␣ IN TROUT
3136
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.
Discussion
Degenerate primer-based PCR is a powerful tool for identifying
related genes in distant species. We used this approach to clone V
domains from rainbow trout which resulted in the subsequent isolation of a CD8␣ homologue from this species. Comparison of
FIGURE 3. A, Genomic sequence of the trout CD8␣ locus. Coding nucleotides are in uppercase and noncoding nucleotides are in lower case
(with the exception of stem loops) for the trout isogenic line OSU-142. The
second methionine (in parentheses) conforms with the preferred Kozak’s
box. Codons found in the cDNA sequence which are split by introns are
underlined and the splice donor (gt) and acceptor (ag) sites are in bold at
the intron borders. Putative transcription factor binding sites are underlined
with the factor identified above. The 30-bp repetitive element (catt. . . tacc)
found in intron 4 is underlined and each of the six copies are designated
with a bracketed number. Primers used to amplify the trout CD8␣ genomic
locus are in bold uppercase at the locus boarders. B, Comparison of the
trout, human, and murine CD8␣ genomic structure. Exons are in roman
numerals with the number of encoded amino acids in parentheses; intronic
distances (in kbp) are found above the introns.
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FIGURE 4. Trout CD8␣ is a single copy gene. Southern blot analysis of
gDNA from four individual animals digested with three different restriction
enzymes. The blot was then probed with the IgSf variable region of trout
CD8␣. One to three bands per digest can be observed consistent with the
presence of a single locus for trout CD8␣. HindIII and EcoRV digests
suggest possible allelic variants (individuals 1 and 2).
The Journal of Immunology
3137
FIGURE 6. Comparison of the MHC class I ␣3 solvent-exposed loop of
HLA and trout that is implicated in CD8␣ binding. Accession numbers are
as follows: HLA-A.28 (P01891), Onmy-UAA*HC-01 (AF115519), and
Onmy-UCA*C32 (U55380).
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
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FIGURE 5. A–C, Expression of trout CD8␣. A, Northern blot analysis
of trout total RNA from indicated tissues probed with the IgSf V domain
of trout CD8␣. A strong signal is observed for the thymus, followed by
weaker transcript levels in the spleen, pronephros, intestine, and mesonephros. B, RT-PCR detection of trout CD8␣ transcripts. Primers located in
exons 1 and 2 of trout CD8␣ were used for PCR amplification of firststrand cDNAs from various tissues (thymus diluted 1:10). Transcripts of
the appropriate size (402 bp) were detected within the thymus, kidney (proand mesonephros), PBLs, spleen, and intestine. Extremely weak signals
were observed for the testis and heart. C, Trout CD8␣ is expressed on the
surface of COS-7 cells transfected with pFlagOmCD8␣. Mock-tranfected
cells were negative for Flag surface expression. Dotted line/shaded area,
M2 anti-Flag/goat anti-mouse IgG1 FITC; and solid line/clear area, goat
anti-mouse IgG1 FITC alone.
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 ␤2 and ␤3 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 ␤2m
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 ␤2m 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 ␤2m 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 CD8␣␤, 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).
Interestingly, the trout CD8␣ 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␣
3138
Acknowledgments
We thank Paul Kincade, Louis Du Pasquier, and Susan Gilfillan for their
suggestions in regard to this manuscript.
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residues thought to be critical for lck dissociation. Transgenic mice
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cell development and activation.
CD8␣ IN TROUT
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