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The Structure of Avian CD5 Implies a Conserved
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J Immunol 1998; 160:4943-4950; ;
http://www.jimmunol.org/content/160/10/4943
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The Journal of Immunology is published twice each month by
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Copyright © 1998 by The American Association of
Immunologists All rights reserved.
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Riitta Koskinen, Thomas W. F. Göbel, Clive A. Tregaskes, John
R. Young and Olli Vainio
The Structure of Avian CD5 Implies a Conserved Function1
Riitta Koskinen,2* Thomas W. F. Göbel,† Clive A. Tregaskes,‡ John R. Young,‡
and Olli Vainio*†
M
ammalian CD5 is a single-chain 67-kDa transmembrane glycoprotein containing three scavenger receptor cysteine-rich (SRCR)3 domains (1, 2). It is found
on all T cells, but expression on B cells differs between species.
Subpopulations of mouse and human B cells, designated B-1a
cells, express CD5, whereas all rabbit peripheral B cells are CD5
positive (3– 6). However, CD5 is not detectable on B cells in rats
or the amphibian Xenopus laevis (7, 8).
During evolution, mammalian CD5 has remained highly conserved, suggesting a role in lymphocyte development and function
(9, 10). Recently, a novel N-glycosylation-dependent ligand for
CD5 has been found on murine splenocytes that is distinct from the
earlier described ligand CD72 (11, 12). Several studies indicate
that CD5 participates in signal transduction involving phosphorylation of intracellular substrates (13, 14), activation of protein kinase C (PKC) (15), and increase in intracellular Ca21 concentration (16). CD5-deficient mice, however, have normal peripheral T
cell responses but markedly increased proliferative capacity of thymocytes (17, 18). Studies on CD5-deficient mice show that CD5
functions as a negative regulator of both thymocyte differentiation
and B cell receptor-mediated signaling in B-1a cells (18, 19).
B-1a cells develop early in ontogeny both in mice and humans
and predominate in the fetal omentum (4, 20, 21). In adults, they
are found in mouse peritoneum and human peripheral blood and
*Turku Immunology Centre and Department of Medical Microbiology, Turku University, Turku, Finland; †Basel Institute for Immunology, Basel, Switzerland; and
‡
Institute for Animal Health, Compton, United Kingdom
Received for publication January 21, 1998. Accepted for publication February 4,
1997.
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 by the Academy of Finland and Turku University Foundation. O.V. was supported by Grant RG366/96 from the Human Frontier Science
Program. Basel Institute for Immunology was founded and is fully supported by F.
Hoffmann-La Roche et Co., Basel, Switzerland.
2
Address correspondence and reprint requests to Dr. Riitta Koskinen, Turku Immunology Centre and Department of Medical Microbiology, Turku University, Kiinamyllynkatu 13, 20520 Turku, Finland.
3
Abbreviations used in this paper: SRCR domains, scavenger receptor cysteine-rich
domains; CLL, chronic lymphocytic leukemia; Ed, embryonic day; ORF, open reading frame; CK2, casein kinase II; PKC, protein kinase C; nt, nucleotide.
Copyright © 1998 by The American Association of Immunologists
lymphoid organs. B-1a cells are self-replenishing and produce low
affinity, polyreactive Abs that may contribute to the development
of autoimmune diseases (22). The number of B-1a cells is increased in most patients with chronic lymphocytic leukemia
(CLL), as well as in patients with HIV infection (23–25).
Here we report the cloning of chicken CD5. The first identification and analysis of a non-mammalian CD5 gene revealed high
homology of the three extracellular SRCR domains and the cytoplasmic region, including potential functional motifs. Furthermore,
using the CD5-specific mAb 2-191, differential expression on ab
vs gd T cells and low CD5 expression on virtually all B cells is
demonstrated. Taken together, the data imply an important role for
CD5 in T and B cell differentiation and function.
Materials and Methods
Animals
Chickens were the H.B2 strain from the Department of Medical Microbiology, Turku University, Turku, Finland, and the H.B19 strain from the
Basel Institute for Immunology Chicken Facility at Gipf-Oberfrick, Switzerland. For ontogenetic studies, fertilized eggs were incubated at 38°C in
80% humidity and the embryonic age was determined by the length of the
incubation period.
Antibodies
For the production of mAb 2-191 (all the mAb were of IgG1 isotype unless
otherwise stated), 2-176, 3-58, and 3-64 BALB/c mice were immunized
three times within 10 days by s.c. injections of either embryonic day 15
(Ed15) thymocytes (2-191 and 2-176) or with a mixture of young adult
PBL and thymocytes (3-64 and 3-58). Fusions and screening of the resulting hybridomas were carried out as previously described (26). mAb 2-6 and
3-298 (IgG2b) recognize chicken CD4 and CD8a, respectively (27, 28).
mAb L22 and 11G2 detect the Bu-1a and Bu-1b molecules, respectively
(29). mAb TCR1 (gd TCR), TCR2 (Vb1TCR), TCR3 (Vb2TCR), and CT3
(CD3) were purchased from Southern Biotechnology Associates
(Birmingham, AL).
Cloning and sequencing of the cDNA
A cDNA library was constructed from thymocyte (8-wk-old H.B2 chicken)
mRNA as described (30). Briefly, the pCDM8 vector (Invitrogen Corporation, San Diego, CA) was digested twice with BstX restriction enzyme
(New England Biolabs, Beverly, MA), and purified from the gel using a
GeneClean kit (Bio 101, Vista, CA). Messenger RNA was prepared using
the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX)
followed by oligo(dT) cellulose chromatography. cDNA synthesis was carried out from 7 mg of the mRNA using the Bethesda Research Lab kit
0022-1767/98/$02.00
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The chicken CD5 cDNA was isolated by COS cell expression cloning utilizing a novel mAb 2-191. The cDNA contains a 1422nucleotide open reading frame encoding a mature protein with 32% and 30% identity to mouse and human CD5 polypeptides,
respectively. The molecule consists of a 330-amino acid extracellular region with three repeats of the scavenger receptor cysteinerich domain, a 29-amino acid hydrophobic transmembrane domain, and a 93-amino acid cytoplasmic tail. The cytoplasmic region
contains motifs that are highly conserved between species, including several potential phosphorylation sites. The chicken CD5 is
a 64-kDa phosphorylated glycoprotein with a protein core of 57 kDa as determined by immunoprecipitation and SDS-PAGE
analysis. ab T cells express a homogeneously high level of CD5, whereas low or intermediate CD5 expression on gd T cells depends
on their tissue location. In contrast to human and mouse, CD5 is found at low levels on all chicken B cells. The high conservation
of structural features, as well as signaling motifs, implies a conserved role for CD5 both in lymphocyte development and
function. The Journal of Immunology, 1998, 160: 4943– 4950.
4944
Biochemical analysis
MDCC-Cu24 cells (a gift from Dr. T. Schat, Cornell University, Ithaca,
NY) (5 3 107) were iodinated with 1 mCi of Na125I (Amersham, Arlington
Heights, IL) by the lactoperoxidase catalyzed reaction essentially as described by Jürgens et al. (8). Postnuclear supernatants were immunoprecipitated by a solid phase method (SPIT), solubilized in Laemmli sample
buffer (LSB), nonreduced without or reduced with 5% 2-ME, and analyzed
on 7% linear SDS-PAGE or 5 to 15% gradient SDS-PAGE.
For glycanase treatments the immunoprecipitates were eluted with 0.5%
SDS, 0.1 M 2-ME for 2 min at 80°C and incubated with 50 U/ml Nglycanase (all glycanase enzymes from Genzyme, Boston, MA) overnight
at 37°C. The pH was changed to 6.3 with 1 M acetic acid before incubation
with 3 U/ml neuraminidase for 2 h at 37°C and overnight incubation with
82 mU/ml O-glycanase. Reduced samples were electrophoresed in
63 LSB.
For 32P-labeling, MDCC-Cu24 cells (5 3 107) were starved for 2 h in
phosphate-free RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 2% BSA. They were labeled with 1.25 mCi/ml HCl-free
[32P]orthophosphate (Amersham) for 3 h at 37°C. The cells were lysed in
buffer containing additional phosphatase inhibitors (0.1 mM Na3Vo4, 0.4
mM EDTA, 10 mM Na4P2O7, 10 mM NaF, and 0.1% NaN3).
Metabolic labeling and immunoprecipitation of transfected COS-7 cells
were carried out as described (32). COS-7 cells were transfected either with
p2.191 or as a control with p2.6 plasmid containing chicken CD4-specific
cDNA (R. Koskinen et al., manuscript in preparation). Preclearing was
performed with a mixture of mAb L22 and 11G2 and immunoprecipitation
with mAb 2-191 or 2-6.
Stable transfection
For stable transfection the CD5 cDNA was subcloned into pCDNA3 plasmid containing a neomycin resistance gene (Invitrogen). Mouse L cells
(3 3 105) were preincubated on a six-well plate (Costar, Cambridge, MA)
in 1 ml DMEM and 10% FCS for 24 h. Plasmid DNA (5 mg) was mixed
with 20 ml lipofectin (Lipofectamine, Life Technologies) in a total volume
of 100 ml H2O. After 30-min incubation at room temperature, 800 ml OptiMEM medium (Life Technologies) was added to the mixture. L cells were
gently washed with prewarmed Opti-MEM before the DNA-liposome mixture was overlaid and the cells were incubated at 37°C 5% CO2 for 5 h.
After transfection, 1 ml DMEM containing 20% FCS was added and 24 h
later the medium was replaced with fresh DMEM containing 10% FCS.
The cells were trypsinized at 72 h after transfection and plated at 1:20
dilution into 96-well plates in DMEM, 10% FCS, and 1 mg/ml G418 (Geneticin, Life Technologies). Growing G418-resistant clones were tested for
CD5 expression by flow cytometry using mAb 2-191.
Immunofluorescence analysis
For immunofluorescence analysis, single-cell suspensions from different
tissues were prepared according to standard procedures. The cells were
incubated with mAb, washed, and further incubated with FITC-conjugated
anti-mouse Ig isotype-specific Abs (purchased from Southern Biotechnology Associates). For double staining the cells were washed and blocked
with normal mouse serum before staining with a phycoerythrin-conjugated
mAb. For three-color staining, the cells were washed and incubated with a
biotin-conjugated mAb followed by streptavidin-Tricolor reagent (Caltag
Laboratories, South San Francisco, CA). After staining, viable cells were
analyzed with a FACScan instrument.
Results
Characterization of the cDNA encoding the 2-191 Ag
COS-7 cells were transfected with a chicken thymocyte cDNA
library in pCDM8 vector. The mAb 2-191 recognizing all thymocytes and a subset of peripheral leukocytes was used to screen the
transfected cells. Following three rounds of COS cell transfection
and immunohistochemical staining, a cDNA clone, designated
p2.191, with a 1976-bp insert, was isolated. It contained an open
reading frame (ORF) of 1422 bp between residues 19 and 1440
(Fig. 1). Protein database searches with the deduced amino acid
sequence revealed the highest similarity with mammalian CD5 sequences. The 2-191 Ag was thus identified as the chicken homologue of the CD5 Ag and will be designated as CD5 throughout
this report. Furthermore, immunofluorescence analysis of L cells
stably transfected with p2.191 revealed a strong uniform staining
with the mAb 2-191 as well as with other putative anti-CD5 mAb
3-64, 3-58, and 2-176 as compared with untransfected cells (data
not shown).
In addition to the ORF, CD5 cDNA clone contained an 18nucleotide (nt) 59 untranslated region and a 513-nt long 39 untranslated region including a polyadenylation signal (AATAAA) 15 nt
prior to the poly(A) tail.
Analysis of the CD5 amino acid sequence
The ORF in p2.191 encodes a 474 amino acid protein. It contains
a putative leader sequence (22 residues), as well as extracellular
(330 residues), transmembrane (29 residues), and cytoplasmic domains (93 residues), which were identified by hydrophobicity plot
analysis and sequence comparison (Fig. 2A) (data not shown). The
leader peptide cleavage site was predicted by the rules of von
Heijne (33) and sequence comparison. The calculated m.w. of the
mature protein is 48,758 and the isoelectric point is 8.1. Amino
acid sequence comparison reveals an overall identity of 32% and
30% to mouse and human CD5, respectively. The highest homology is found in the hydrophobic transmembrane region (71% and
74% to mouse and human, respectively), and the cytoplasmic tail
with 51% amino acid identity both to mouse and human CD5.
There are several long stretches of sequence identity between species in the cytoplasmic region of CD5 sequences, including different motifs for phosphorylation (Fig. 2A). PKC has two potential
target sites (SKK at position 368 and TPR at 394) for phosphorylation. The Prosite pattern search revealed a putative tyrosine
phosphorylation site RRGDNDY at position 412 in chicken CD5,
which is also found in mouse and human CD5. Furthermore,
serine-containing conserved motifs (SDSD and SDYD at positions
440 and 442) for casein kinase II phosphorylation, and a nonconserved site (KRIS at position 368) for both cAMP- and cGMPdependent protein kinase, were also present in the cytoplasmic
region of chicken CD5.
The extracellular domain, however, is less well conserved (26%
and 22% to mouse and human, respectively). In the chicken CD5,
there are three extracellular SRCR domains that all have the cysteine residues conserved as compared with mammalian CD5 sequences (Fig. 2A). The first and the third SRCR domains contain
eight cysteines (except for the N-terminal domain of the bovine
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(Igress, CA). The first strand synthesis was primed with an oligonucleotide:
59-AACCCGGCTCGAGCGGCCGCT(18)-39. The cDNA was size fractionated by electrophoresis, and regions of 1 to 2 kb and 2 to 5 kb were
excised from the gel and purified. The cDNA mixture from both purifications was ligated into BstX I cut pCDM8 vector using phosphorylated BstX
I adapters (Invitrogen). The numbers of colonies obtained were 3.6 3 106
from the 1- to 2-kb cDNA and 1.8 3 106 from the 2- to 5-kb cDNA.
Plasmid purification was conducted according to the standard
protocols (31).
COS-7 cells (0.5 3 106) were transfected with 3 mg of the cDNA library
in a HEPES-buffered DMEM solution containing 500 mM DEAE-dextran
and 100 mM chloroquine as described (32). The transfection mixture was
incubated at 37°C for 2 h, washed, and cultured on a chamber slide in
bicarbonate-buffered DMEM containing 10% FCS. After 48 h, the cells
were fixed with acetone and immunostained with 2-191 mAb. A singlepositive cell was picked under a microscope with a Drummond sequencing
pipet (Drummond Scientific Company, Broomall, PA) into 100 ml of Hirt
extraction solution (10 mM EDTA, 0.6% SDS, and 100 mg/ml proteinase
K) to further isolate plasmids by a method described earlier (30).
The isolated plasmids were electroporated into MC1061/P3 bacteria
(Invitrogen), plasmid DNA was prepared (QIAprep Spin Plasmid Miniprep
kit, Qiagen, Chatsworth, CA), and a positive clone p2.191 was isolated by
screening successively smaller pools of plasmids. Double-stranded sequencing was carried out on both strands using a Sequenase kit, version 2.0
(United States Biochemical, Cleveland, OH). Sequence data were analyzed
by the GCG (Genetics Computer Group, Madison, WI), Lasergene Molecular Biology Software (DNASTAR, Madison, WI), and Prosite pattern
search (EMBL, Heidelberg, Germany) programs.
CHICKEN CD5
The Journal of Immunology
4945
vertebrate, the sea lamprey Petromyzon marinus (34). The deduced protein consists of two SRCR domains flanking five epidermal growth factor-like repeats. Homology analysis was performed to determine whether chicken CD5 SRCR domains are
more closely related to those of the primitive or modern vertebrates. The analysis showed that the first two N-terminal domains of chicken CD5 were 15 to 20% homologous to SREG
and 14 to 28% to mammalian domains (Fig. 2B) (data not
shown). The corresponding homology figures for the third domain were 30 to 34% and 18 to 20% to mammalian and SREG
SRCR domains, respectively. However, the phylogenetic analysis showed that the SRCR domains cluster together and did not
indicate that the chicken domains are closer to SREG SRCR
than mammalian SRCR domains (data not shown).
Biochemical analysis of CD5
Ontogeny and tissue distribution of chicken CD5
FIGURE 1. The nucleotide and the deduced amino acid sequence of
chicken CD5. The extracellular cysteines are in bold type. Putative Nglycosylation sites, the transmembrane region, and the polyadenylation signal are underlined. These sequence data are available from EMBL under
accession number Y12011.
CD5 which has only six), whereas the second SRCR domain contains six cysteines. This domain organization is a typical feature of
CD5 molecules within the family of SRCR proteins (2). In the
mammalian CD5 sequence a threonine- and proline-rich sequence
is thought to form an extended peptide stretch separating the first
two SRCR domains (1, 3). This is also found in chicken CD5
(residues 101–121) (Fig. 2A). The extra sequence in the N terminus of the rat CD5 is not present in the chicken sequence. Two
potential N-glycosylation sites are located in the first SRCR and a
third site is found in the second SRCR.
A cDNA encoding a SREG protein, a putative type I integral
membrane protein, has been isolated from the most primitive
During ontogeny, CD5 was first detected at Ed10 on about 5% of
thymocytes (Fig. 4A). The frequency of CD5-positive cells rapidly
increased during the next few days of embryonic development to
reach adult levels at around Ed16. Three-color immunofluorescence analysis of adult thymocytes indicated that about 50% of the
CD4/CD8 double-negative thymocytes already expressed CD5
(Fig. 4B). Virtually all CD4/CD8 double-positive, as well as single-positive, thymocytes expressed CD5 with increasing fluorescence intensity.
More than 90% of thymocytes, as well as most bursa cells
from a 3-wk-old animal, expressed CD5 uniformly (Fig. 5).
However, the CD5 intensity on bursa cells was substantially
lower than on thymocytes (Fig. 5). Interestingly, CD5 was also
expressed on most PBL and spleen cells with two distinct subpopulations having different fluorescence intensities. High levels of CD5 expression were found on the majority of the lymphocytes, whereas a distinct subset displayed lower CD5
fluorescence intensity.
Two-color immunofluorescence analysis of PBL demonstrated that CD5 was expressed on virtually all CD31 T cells
(Fig. 6A). Both ab T cell subpopulations expressing either Vb1
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Immunoprecipitation and SDS-PAGE analysis of an iodinated T
cell lysate identified the CD5 Ag as a 64-kDa monomeric protein,
both under reducing and nonreducing conditions (Fig. 3A) (data
not shown). N-glycanase treatment revealed a protein core of 57
kDa, whereas neither neuraminidase nor O-glycanase treatments
significantly changed the migration in the gel (Fig. 3B). Since
mammalian CD5 has been shown to be a phosphoprotein (35), we
performed a metabolic labeling with [32P]orthophosphate. A 64kDa protein was precipitated with two CD5-specific mAb 2-191
and mAb 3-64 (Fig. 3C) showing that chicken CD5 is
phosphorylated.
To determine whether T cell activation induces tyrosine phosphorylation of chicken CD5 we stimulated MDCC-Cu24 cells with
PMA or pervanadate followed by specific immunoprecipitation
and a phosphotyrosine blot analysis. We were unable to detect any
tyrosine phosphorylation of the molecule (data not shown), suggesting that the phosphorylation of chicken CD5 is due to serine
and/or threonine phosphorylation.
To provide further evidence that p2.191 clone encodes CD5 we
transfected COS cells transiently with p2.191, and labeled the cells
metabolically. A 64-kDa band was specifically immunoprecipitated from the lysed transfected COS cells (Fig. 3D). This demonstrates that p2.191-transfected COS cells produce a full length
glycosylated CD5 protein. In conclusion, chicken CD5 is a 64-kDa
monomeric, phosphorylated glycoprotein.
4946
CHICKEN CD5
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FIGURE 2. A, Amino acid alignment of CD5 sequences. The mature chicken CD5 sequence is compared with the corresponding mammalian sequences,
using the Clustal method and the PAM250 residue weight table. Shaded amino acids represent residues in which at least residues from two species match
the chicken sequence. The cysteine residues are numbered according to their position in the SRCR motifs. Residues forming potential signaling motifs are
marked with a star (see text for details). The bar indicates the transmembrane region. B, Amino acid alignment of the three SRCR domains of chicken CD5
and the two SRCR domains of the sea lamprey, P. marinus, SREG protein. Shaded residues match one or both of the SREG SRCR domain residues.
or Vb2 family receptors displayed high CD5 levels, whereas the
CD5 expression was markedly lower on all gd T cells (Fig. 6B).
About 75% of splenic gd T cells had an intermediate level and
approximately 25% exhibited low CD5 expression levels (Fig.
6C). An opposite staining pattern was observed in peripheral
blood gd T cells. The intermediate CD5 expression correlated
The Journal of Immunology
4947
with the expression of CD8 on gd T cells (data not shown). In
addition, T cell stimulation by mitogens or anti-CD28 mAb
up-regulates the surface expression of CD5 as analyzed by flow
cytometry (Fig. 7) (data not shown).
FIGURE 4. A, Ontogeny of CD5 in thymus.
Thymocytes from H.B2 strain embryos at different time points were isolated and stained for surface expression of CD5 by mAb 2-191, and analyzed by flow cytometry. B, The expression of
CD5 on thymocytes. Three-color analysis of CD5
on thymocyte subpopulations defined by CD4
and CD8.
All peripheral B cells, as detected by Bu-1 Ag expression, were
also CD5 positive (Fig. 6A). The CD5 fluorescence intensity on B
cells was comparable with that of the intermediate CD5-positive
gd T cells.
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FIGURE 3. Biochemical analysis of chicken CD5. MDCC-Cu24 cells were labeled either with 125I (A and B) or 32P (C). The lysates were immunoprecipitated with mAb 2-191 (A–C) and 3-64 (C) and further treated with glycanases as indicated (B). Samples were analyzed under reducing conditions
by SDS-PAGE on 5 to 15% gels. The control precipitation (C) was performed with an isotype-matched mAb with irrelevant specificity. The migration of
m.w. standards is indicated to the right. Immunoprecipitation of 35S-metabolically labeled (D) pCD5 and pCD4 (control plasmid-encoding chicken
CD4)-transfected COS cells. The samples were immunoprecipitated with 2-191 or 2-6 mAb and analyzed on 12% SDS-PAGE under reducing conditions.
The migration of m.w. standards is indicated to the left.
4948
CHICKEN CD5
FIGURE 5. Tissue distribution of CD5. The expression of CD5 in thymus, bursa, spleen, and peripheral blood of a 3-wk-old H.B19 bird was analyzed by
flow cytometry. Cells were stained with mAb 2-191
and with an isotype-matched control Ab (faint line).
CD5 belongs to a protein family characterized by multiple repeats of the extracellular SRCR domains. Each member of the
SRCR family has a specific domain organization with typically
conserved cysteine residues (2). CD5 is composed of three
SRCR domains like CD6, whereas WC1 (expressed on bovine
CD42, CD82, gd T cells) includes 11 domains (2). The SRCR
domains of chicken CD5 are structurally very similar to the
mammalian homologues with identical cysteine residue composition and location. The structural similarity of the CD5 molecules is strengthened by finding also in chicken CD5 a proline/
threonine-rich region separating SRCR domains one and two
(Fig. 2A). This region is supposed to form an extended hinge
separating the first two domains, and its conservation in evolution points to its importance (1, 3).
Chicken CD5 represents the first non-mammalian CD5 that
has been cloned. Therefore, we studied whether chicken domains have ancient SRCR features or are closer to the mammalian domains. Interestingly, chicken CD5 SRCR domains are
as homologous to the sea lamprey SREG protein SRCR domains (34) as to mammalian CD5 SRCR domains. However, the
phylogenetic analysis indicated that chicken CD5 domains bear
more similarity to other members of the CD5 subfamily of
SRCR domains than to the lamprey SREG protein SRCR domains. These findings suggest that the appearance of CD5 has
preceded the split during evolution of avian and mammalian
branches, although the SRCR domain as a building block of a
membrane protein is an ancient one.
The 93-amino acid long cytoplasmic tail of the chicken CD5
contains several conserved motifs potentially involved in signal
transduction (Fig. 2A). These motifs include phosphorylation
sites for kinases that are able to phosphorylate tyrosine or
serine/threonine residues. Chicken, mouse, and human CD5
have a conserved threonine residue at position 394 for possible
phosphorylation by PKC. This is the only target for PKC in
mouse and human, however, the chicken sequence contains an
additional potential PKC phosphorylation site at position 368.
The lack of tyrosine phosphorylation of chicken CD5 following
T cell activation suggests that the phosphorylation pattern differs from that of mammals (13).
There are two highly conserved adjacent phosphorylation sites
for CK2 at the end of the cytoplasmic tail of CD5. Furthermore,
mouse and human CD5 sequences have additional sites for phosphorylation by CK2. CK2 is found in high concentrations in transformed cells (36), and thus the potential phosphorylation of CD5
by CK2 may be of importance in the development of the CD51 B
cell malignancy CLL.
During ontogeny the first CD5-positive cells both in the thymus
and bursa are detected on Ed10, about 2 to 3 days before the first
TCR and surface Ig expression. By immunofluorescence analysis
CD5 was observed on about half of the leukocytes in Ed13 bone
marrow (data not shown). This indicates that CD5 expression on T
or B cell precursors starts before they migrate into the primary
lymphoid organs. By the time functional TCR and Ig gene rearrangements and their expression have been completed, all T and B
cells in the primary lymphoid organs express CD5. Peripheral ab
T cells express a relatively high level of CD5 whereas a much
lower level of the CD5 Ag is detected on gd T cells and B cells.
The control of CD5 expression on T cells is activation dependent,
since activated T cells have up-regulated CD5 expression. This
may explain the variable CD5 expression on gd T cells, as the
CD8-bearing gd T cells that have a higher CD5 expression than
CD8-negative gd T cells are also in an activated stage ex vivo
(express MHC class II and IL-2R) (37). The overall low CD5
expression on gd T cells may imply a different need for coreceptor
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Discussion
The Journal of Immunology
4949
and signaling molecules in their activation as compared with ab T
cells. In chicken, this is exemplified by the stable expression of gd
TCR during differentiation in the thymus and by the lack of CD28
on most peripheral gd T cells (38). Altogether this may indicate a
more crucial role for CD5 in ab T cell activation.
The adaptive immune systems of birds and mammals are very
similar. Avian B cells, however, are physiologically distinct
from their mammalian counterparts. They are generated during
a limited period of time in the bursa of Fabricius and the diversity of Ig genes is generated by a process of segmental gene
conversion (39). After involution of the bursa of Fabricius, the
peripheral pool of B cells is replenished from a self-renewable
peripheral B cell population (40). In contrast, the majority of
human and mouse B cells derive from progenitor cells throughout life, first in fetal liver and then in the bone marrow. Their
Ig diversity is generated by gene rearrangement and somatic
hypermutation (39). However, the small subset of B-1a cells
bear close resemblance to chicken B cells, being generated only
during early development (20). Chicken B cells as well as B-1a
FIGURE 7. The expression of CD5 on unstimulated and ConA-stimulated peripheral blood lymphocytes from a 3-wk-old H.B2 bird.
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FIGURE 6. The expression of CD5 on T and B cells. The peripheral CD5 expression was analyzed by flow cytometry on PBL (A–C) and spleen cells
(C) of a 3-wk-old H.B19 animal using the mAbs as shown. Percentage of positive cells is indicated in the appropriate quadrants.
4950
cells in mammals express CD5, which may indicate that all
chicken B cells are developmentally equivalent to B-1a cells
and CD5 has a role in the differentiation and renewal of this
type of B cells.
The rabbit is an exception to mammals so far studied in that all
its B cells are CD5 positive (6). Thus it seems unlikely that this
feature, CD5-bearing self-renewing peripheral B cells, would be
an ancient phenomenon. Instead, it is rather a highly specialized
feature that developed independently in rabbits and chickens by
convergency and was not a feature of the common ancestor of
birds and mammals.
The analysis of chicken CD5 presented here reveals conservation of the structural features of the extracellular SRCR domains,
as well as in long stretches of the cytoplasmic sequence. The fact
that the cytoplasmic domain of CD5 contains amino acid motifs
being highly conserved between birds and mammals suggests an
important function for CD5 in the control of lymphocyte differentiation and activation.
We thank Drs. Kerry Campbell, Beat Imhof, and Paavo Toivanen for critical reading of the manuscript; Drs. Jari Jalava, Mikael Skurnik, and Tatsuya Uchida for advice in sequence analysis; and Dr. Ton Schat for MDCC
cell line. Aija Kaitaranta, Marjo Hakkarainen, and Barbara Ecabert are
acknowledged for their excellent technical assistance.
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Acknowledgments
CHICKEN CD5