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The Journal of Immunology, 1998, 160: 4943-4950.
Copyright © 1998 by The American Association of Immunologists

The Structure of Avian CD5 Implies a Conserved Function1

Riitta Koskinen2,*, Thomas W. F. Göbel{dagger}, Clive A. Tregaskes{ddagger}, John R. Young{ddagger} and Olli Vainio*,{dagger}

* Turku Immunology Centre and Department of Medical Microbiology, Turku University, Turku, Finland; {dagger} Basel Institute for Immunology, Basel, Switzerland; and {ddagger} Institute for Animal Health, Compton, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The chicken CD5 cDNA was isolated by COS cell expression cloning utilizing a novel mAb 2-191. The cDNA contains a 1422-nucleotide 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 cysteine-rich 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. {alpha}ß T cells express a homogeneously high level of CD5, whereas low or intermediate CD5 expression on {gamma}{delta} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mammalian 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, 4, 5, 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 Ca2+ 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 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, 24, 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 {alpha}ß vs {gamma}{delta} 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 CD8{alpha}, respectively (27, 28). mAb L22 and 11G2 detect the Bu-1a and Bu-1b molecules, respectively (29). mAb TCR1 ({gamma}{delta} TCR), TCR2 (Vß1TCR), TCR3 (Vß2TCR), 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 µg of the mRNA using the Bethesda Research Lab kit (Igress, CA). The first strand synthesis was primed with an oligonucleotide: 5'-AACCCGGCTCGAGCGGCCGCT(18)-3'. 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 x 106 from the 1- to 2-kb cDNA and 1.8 x 106 from the 2- to 5-kb cDNA. Plasmid purification was conducted according to the standard protocols (31).

COS-7 cells (0.5 x 106) were transfected with 3 µg of the cDNA library in a HEPES-buffered DMEM solution containing 500 µM DEAE-dextran and 100 µM 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 single-positive cell was picked under a microscope with a Drummond sequencing pipet (Drummond Scientific Company, Broomall, PA) into 100 µl of Hirt extraction solution (10 mM EDTA, 0.6% SDS, and 100 µg/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.

Biochemical analysis

MDCC-Cu24 cells (a gift from Dr. T. Schat, Cornell University, Ithaca, NY) (5 x 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 N-glycanase (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 6x LSB.

For 32P-labeling, MDCC-Cu24 cells (5 x 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 x 105) were preincubated on a six-well plate (Costar, Cambridge, MA) in 1 ml DMEM and 10% FCS for 24 h. Plasmid DNA (5 µg) was mixed with 20 µl lipofectin (Lipofectamine, Life Technologies) in a total volume of 100 µl H2O. After 30-min incubation at room temperature, 800 µl Opti-MEM 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go). 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).



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  		FIGURE 1. The nucleotide and the deduced amino acid sequence of chicken CD5. The extracellular cysteines are in bold type. Putative N-glycosylation sites, the transmembrane region, and the polyadenylation signal are underlined. These sequence data are available from EMBL under accession number Y12011.

 
In addition to the ORF, CD5 cDNA clone contained an 18-nucleotide (nt) 5' untranslated region and a 513-nt long 3' 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. 2GoA) (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. 2GoA). 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 cGMP-dependent protein kinase, were also present in the cytoplasmic region of 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.

 
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. 2GoA). The first and the third SRCR domains contain eight cysteines (except for the N-terminal domain of the bovine 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. 2GoA). 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 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. 2GoB) (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

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. 3GoA) (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. 3GoB). Since mammalian CD5 has been shown to be a phosphoprotein (35), we performed a metabolic labeling with [32P]orthophosphate. A 64-kDa protein was precipitated with two CD5-specific mAb 2-191 and mAb 3-64 (Fig. 3GoC) showing that chicken CD5 is phosphorylated.



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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.

 
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. 3GoD). 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.

Ontogeny and tissue distribution of chicken CD5

During ontogeny, CD5 was first detected at Ed10 on about 5% of thymocytes (Fig. 4GoA). 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. 4GoB). Virtually all CD4/CD8 double-positive, as well as single-positive, thymocytes expressed CD5 with increasing fluorescence intensity.



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  		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.

 
More than 90% of thymocytes, as well as most bursa cells from a 3-wk-old animal, expressed CD5 uniformly (Fig. 5Go). However, the CD5 intensity on bursa cells was substantially lower than on thymocytes (Fig. 5Go). 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.



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  		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).

 
Two-color immunofluorescence analysis of PBL demonstrated that CD5 was expressed on virtually all CD3+ T cells (Fig. 6GoA). Both {alpha}ß T cell subpopulations expressing either Vß1 or Vß2 family receptors displayed high CD5 levels, whereas the CD5 expression was markedly lower on all {gamma}{delta} T cells (Fig. 6GoB). About 75% of splenic {gamma}{delta} T cells had an intermediate level and approximately 25% exhibited low CD5 expression levels (Fig. 6GoC). An opposite staining pattern was observed in peripheral blood {gamma}{delta} T cells. The intermediate CD5 expression correlated with the expression of CD8 on {gamma}{delta} 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. 7Go) (data not shown).



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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.

 


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  		FIGURE 7. The expression of CD5 on unstimulated and ConA-stimulated peripheral blood lymphocytes from a 3-wk-old H.B2 bird.

 
All peripheral B cells, as detected by Bu-1 Ag expression, were also CD5 positive (Fig. 6GoA). The CD5 fluorescence intensity on B cells was comparable with that of the intermediate CD5-positive {gamma}{delta} T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 CD4-, CD8-, {gamma}{delta} 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. 2GoA). 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. 2GoA). 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 CD5+ 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 {alpha}ß T cells express a relatively high level of CD5 whereas a much lower level of the CD5 Ag is detected on {gamma}{delta} 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 {gamma}{delta} T cells, as the CD8-bearing {gamma}{delta} T cells that have a higher CD5 expression than CD8-negative {gamma}{delta} T cells are also in an activated stage ex vivo (express MHC class II and IL-2R) (37). The overall low CD5 expression on {gamma}{delta} T cells may imply a different need for coreceptor and signaling molecules in their activation as compared with {alpha}ß T cells. In chicken, this is exemplified by the stable expression of {gamma}{delta} TCR during differentiation in the thymus and by the lack of CD28 on most peripheral {gamma}{delta} T cells (38). Altogether this may indicate a more crucial role for CD5 in {alpha}ß 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 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.


   Acknowledgments
 
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.


   Footnotes
 
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. Back

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. Back

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. Back

Received for publication January 21, 1998. Accepted for publication February 4, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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