Molecular characterization of CD28 and

cytotoxic T-lymphocyte-associated

antigen 4 (CTLA-4) of woodchuck

(Marmota monax)

D. Yang

M. Roggendorf

M. Lu

Authors’ affiliations:

D. Yang,

M. Roggendorf,

M. Lu

Institut fur Virologie,

Universitatsklinikum Essen,

Essen, Germany

Correspondence to:

Dr Mengji Lu

Institut fur Virologie

Universitatsklinikum Essen

Hufelandstrasse 55

D-45122 Essen

Germany

Tel:þ49 7233530

Fax:þ49 7235929

e-mail: mengji.lu@

uni-essen.de

Abstract: Eastern woodchuck (Marmota monax) became an important

animal model to study the immunological processes in hepatitis B virus

infection. To facilitate further study of T-cell responses in this model, we

cloned and sequenced the cDNAs of Woodchuck CD28 and cytotoxic

T-lymphocyte-associated antigen 4 (CTLA-4), which play important roles for

the regulation of T-cell activation by delivering the costimulation signals.

According to the deduced amino-acid sequences, Woodchuck CD28 showed a

similarity of 70% to 80% to its mammalian homologues. Woodchuck CTLA-4

has a higher similarity of 74% to 85% to corresponding mammalian CTLA-4

molecules. The strict conservation of critical amino-acid residues like cystein

and asparagine residues in Woodchuck CD28 and CTLA-4 suggests that both

molecules may structurally resemble their human or mouse homologues. A

hexapeptide motif, MYPPPY, which has been supposed to be essential for the

interaction with CD80, is present in both Woodchuck CD28 and CTLA-4. The

cloned cDNAs of Woodchuck CD28 and CTLA-4 were placed under the

control of the cytomegalovirus (CMV) promoter of the mammalian expression

vector pcDNA3. Both proteins were expressed and detected by respective

crossreactive antibodies in transiently transfected mammalian cells. By

immunohistochemical staining with these antibodies, CD28 and CTLA-4

were also detected on cultured woodchuck peripheral blood lymphocytes.

The molecular characterization of Woodchuck CD28 and CTLA-4 will

facilitate studies on the T-cell response to hepadnavirus in the woodchuck

model.

Eastern woodchuck (Marmota monax) is the natural host of wood-

chuck hepatitis virus (WHV), a closely related virus of human hepa-

titis B virus (HBV), and represents a useful animal model to study the

host immune response to acute and chronic HBV infection (1–4). The

T-cell response to WHV in woodchucks is of particular interest

because of its pivotal role in virus clearance of a primary viral

Key words:

accessory molecules; CD28; costimulation;

CTLA-4; woodchuck

Acknowledgments:

We thank Thekla Kemper and Anja Busse for

excellent technical assistance. This work was

support in part by Deutsche

Forschungsgemeinschaft (RO 687/6–1).

Received 7 March 2003, revised 10 April 2003, and accepted

for publication 15 April 2003

Copyright � Blackwell Munksgaard 2003

Tissue Antigens. ISSN 0001-2815

Tissue Antigens 2003 62: 225–232

Printed in Denmark. All rights reserved

The nucleotide sequence data reported in this paper have beenassigned the GenBank accession numbers AF130427 (WoodchuckCD28) and AF130428 (Woodchuck CTLA-4).

225

infection (5–7). One of the important characteristic of chronic

hepadnavirus infection is the lack of proper T-cell responses to

viral proteins (7,8). However, only few relevant components of the

immune system of woodchuck were recently cloned and molecularly

characterized (9–13). Of particular note, no information about the

molecules involved in T-cell activation and regulation is available

to date.

T-cell activation requires at least two signals delivered by

antigen-presenting cells (APCs). Two accessory molecules, CD28

and cytotoxic T-lymphocytes-associated molecule 4 (CTLA-4),

expressed on lymphocytes interact with B7 molecules and regulate

the activation of T cells (for reviews: 14–18). CD28 is constitu-

tively expressed on the cell surface of the majority of resting and

activated T cells (19). The function of CD28 is to deliver a positive

costimulation signal through interaction with B7 molecules on

T cells engaged by the MHC–TCR interaction (19,20). In contrast

to CD28, CTLA-4 has been found to be mainly expressed in low

numbers on activated T cells (21–23). The CTLA-4-Ig fusion

protein has a 10–100-fold higher avidity for CD80 than the

corresponding CD28-Ig fusion protein (24). Currently, the role of

CTLA-4 in the regulation of T-cell activation has been intensely

studied. Blocking CTLA-4 by using Fab fragments of specific

monoclonal antibodies (mAbs) led to clonal expansion of T cells,

while crosslinked anti-CTLA-4 mAbs inhibited T-cell activation

(25–27). Mice with a targeted mutation of CTLA-4 developed fatal

lymphoproliferative disease at 2–4 weeks of age (28, 29). These

experiments suggested that CTLA-4 is a negative regulator for

T-cell activation though alternative interpretations were discussed

(17, 18, 25–27, 30, 31). In signal transduction through CD28 and

CTLA-4, phosphotidylinositol 3 (PI3)-kinase may play a key role

(32–39).

Both CD28 and CTLA-4 molecules are type I transmembrane

glycoproteins and members of the immunoglobulin superfamily

(21, 40). They are expressed on the cell surface of T cells as a

disulfide-linked homodimer (40–42). A well-conserved hexapeptide

sequence motif MYPPPY in CD28 and CTLA-4 was supposed to be

important for binding to the CD80 and CD86 ligands. Other sequence

motifs, YMNMTPR in CD28 and YVKMPP in CTLA-4, are suggested

to play a role for binding of the 85-kDa subunit of phosphotidylino-

sitol 3 (PI3)-kinase (16).

In the present work, we cloned cDNAs of Woodchuck CD28

and CTLA-4 and performed the detection of their expression in

transiently transfected mammalian cells and on cultured wood-

chuck peripheral blood lymphocytes with respective specific

antibodies. The availability of these reagents will facilitate further

studies on the T-cell response to hepadnavirus in the woodchuck

model.

Materials and methods

Culturing of woodchuck peripheral blood lymphocytes (PBLs)

Woodchuck PBLs were separated by Ficoll-Paque (Pharmacia,

Freiburg, Germany) density gradient centrifugation and suspended

in AIM-V medium (Gibco BRL, Eggenstein-leopoldshafen, Germany)

supplemented with 2% 0.2M l-glutamine (Sigma, Deisenhofen,

Germany), 1% 0.125M gentamicin sulfate (Sigma) and 10% fetal

calf serum (Cytokine, Germany). The PBMCs were cultured in a

density of 106 permL in 60-mm well plates (Falcon, Becton Dickinson,

NJ) at 37�C in a humidified atmosphere containing 5%CO2. To

stimulate PBMC proliferation, phytohemagglutinin (Sigma) at a

concentration of 2 mgmL�1 and 50 units of interleukin 2 (R & D

system, Germany) per mL was added.

Reverse transcription, polymerase chain reaction, cloning

and sequencing: Construction of plasmids peWCD28 and

peWCTLA4 for the expression in mammalian cells

cDNA fragments comprising the coding region of Woodchuck CD28

and CTLA-4 were amplified by RT-PCR by using the primers

described in Table 1. The PCR products were cloned into pCRII

vectors (Invitrogen, San Diego, CA) according to the manufacturer’s

instructions. Three clones of each were sequenced to verify the

correct nucleotide sequence. The cloned cDNA fragments of

Woodchuck CD28 and CTLA-4 were isolated by digestion with

HindIII and XhoI and inserted into the corresponding site of

pcDNA3 vector predigested with HindIII and XhoI (Invitrogen). In

generated plasmids peWCD28 and peWCTLA4 the coding regions of

both genes were placed under the control of the CMV promoter of

pcDNA3.

Transient expression of Woodchuck CD28 and CTLA-4 by

transfection of peWCD28 and peWCTLA4 into a baby hamster

kidney (BHK) cell line. Plasmids peWCD28 and peWCTLA4 were

prepared using the plasmid purification kit (Qiagen, Hilden,

Germany). The BHK cells were transfected by using lipofectamine

(Gibco BRL). Four-mg plasmids were incubated with 10 mg of

lipofectamine in 100 mL of Opti-Media (Gibco BRL) for 45min

and was further incubated with cells in 1mL of Opti-Media for

6 h at 37�C, 5%CO2. Transfected cells were maintained for 48 h

at 37�C, 5%CO2 and fixed with 50% methanol. The expressed

Woodchuck CD28 and CTLA-4 were detected by indirect

immunofluorescence staining using goat antisera to conserved

regions of mouse CD28 (amino acid 198–217) and CTLA-4 (amino

acid 205–223), respectively (Santa Cruz Biothechnology, Heidelberg,

Germany).

Lu et al : Woodchucks CD28 and CTLA-4

226 Tissue Antigens 2003: 62: 225–232

Immunohistochemical detection of Woodchuck CD28 and

CTLA-4 on woodchuck peripheral blood lymphocytes

Cultured woodchuck PBLs were spinned down ona slide and immuno-

histochemically stained for CD28 and CTLA-4 molecules by using the

DAKO SLAB kit according to the manufacturer’s instructions. Polyclonal

goat anti-CD28 and anti-CTLA-4 IgG (1 : 40 diluted, Santa Cruz Biotechol-

ogy) were used as primary antibodies. The development of staining was

performed with the New Fuchsin chromogen solution. Nuclei of lympho-

cytes were stained with hematoxylin. Control staining was performed by

replacing the primary antibodies with non-specific goat IgGs.

Results

RT-PCR amplification: Cloning and sequence analysis of

cDNA fragments comprising the coding region of

Woodchuck CD28 and CTLA-4

Using the primers described in Table 1, RT-PCRs were performed with

total RNAs isolated from PHA-activated woodchuck lymphocytes. An

approximately 700-bp long cDNA fragment was visible on the agarose

gel after the nested PCR amplification using CD28p3 and CD28p4.

CD28p3 was designed according the human CD28 cDNA sequence,

because the corresponding primer 50-CAG AAC AAT GAC ACT CAG

GC-30 of CD28p3 according to the mouse CD28 cDNA sequence in

combination with CD28p4 failed to amplify specific PCR products.

Sequencing of cloned PCR products revealed that the amplified cDNA

fragment covers the complete coding region of Woodchuck CD28 (Fig. 1).

The nucleotide sequence of Woodchuck CD28 showed a similarity of 70–

80% to the CD28 sequence of other mammalian species and of 51% to

the chicken CD28 sequence (Table 2). The deduced amino-acid sequence

of the Woodchuck CD28 precursor has a length of 221 amino-acid

residues, and shows the highest similarity of 79% to rabbit CD28

(Fig. 2A). Like other mammalian CD28, the similarity of Woodchuck

CD28 to the chicken CD28 homologue lies at 52%. All cystein residues

and asparagine residues except the N at aa 86 within the extracellular

domain are conserved in Woodchuck CD28, indicating that Woodchuck

CD28 may structurally resemble human and other mammalian CD28

molecules.

The cDNA fragment comprising the coding region of Woodchuck

CTLA-4 was amplified by nested RT-PCR using primers correspond-

ing to mouse CTLA-4 sequences flanking the coding region, which

are well conserved across mammalian species. The cDNA fragment

of Woodchuck CTLA-4 was cloned and sequenced. Woodchuck

CTLA-4 shows a high similarity of 74% to 85% to other mammalian

CTLA-4 at the amino-acid level (Fig. 2B). The putative leader

sequence and extracellular part of the mature protein (aa 1–161) of

CTLA-4 is rather heterogeneous, whereas the transmembrane and

cytoplasmic domains (aa 162–220) are extremely conserved. All

cystein and asparagine residues within the extracellular domain

present in human CTLA-4 are conserved in Woodchuck CTLA-4.

Woodchuck CD28 and CTLA-4 showed a similarity of approxi-

mately 46% to each other at the level of nucleotide sequences (Fig. 1).

A hexapeptide motif MYPPPY is conserved in both Woodchucks

CD28 and CTLA-4, which has been shown to be important

for binding to the CD80 and CD86 ligands. The binding motifs for

the p85 subunit of phosphoinositide 3-kinase, YMNMTPR of CD28

(aa 192–197) and YVKMPP (aa 201–206), are conserved in the

deduced amino-acid sequences of corresponding woodchuck proteins

(16).

Description of primers for polymerase chain reaction amplification of the complete coding sequence of Woodchucks CD28 and CTLA-4.

Polarity Primer Name Position

CD28

RT-PCR Sense 50 -TGC AGC CCT GGC CCT CAT CAG-30 CD28p1 32a

Antisense 50 -AGT GGC CGG CTG AAG ATG AGG CTG-30 CD28p2 813a

Nested PCR Sense 50 -GAC AAA GAT GCT CAG GCT GC-30 CD28p3 93b

Antisense 50 -GCC GGC GGG CTT CTG GAT AG-30 CD28p4 742a

CTLA-4

RT-PCR Sense 50 -GAT CCT GTT GGG TTT TAC TC-30 CTLA4p1 1c

Antisense 50 -GCC TCA GCT TTT AGA GAC TG-30 CTLA4p2 781c

Nested PCR Sense 50 -AGC CAT GGC TTG TCT TGG ACT CC-30 CTLA4p3 54c

Antisense 50 -TTT CAG TTG ATG GGA ATA AAA TAA GG-30 CTLA4p4 731c

aAccession number M34563 (mouse).bAccession number J02988 (human).cAccession number X05719 (mouse).

Table 1

Lu et al : Woodchucks CD28 and CTLA-4

Tissue Antigens 2003: 62: 225–232 227

Expression of Woodchuck CD28 and CTLA-4 in mammalian

cells by transient transfection and detection by indirect

immunofluorescence staining

The BHK cells were transfected with plasmids peCD28 and peCTLA4

and stained with goat anti-CD28 and anti-CTLA-4 antibodies, respec-

tively. These polyclonal antibodies were directed to the conserved

regions of mouse CD28 (amino acid 198–217) and CTLA-4 (amino

acid 205–223). In the region of aa 198–217 to that of CD28, Wood-

chuck CD28 differs from mouse CD28 by three amino-acid residues at

positions 202, 203, and 205. The region aa 205–223 of CTLA-4 is

identical in all species characterized to date. Thus, the antibodies to

the conserved region of both molecules are crossreactive to Wood-

chucks CD28 and CTLA-4. Positive staining of transfected cells was

clearly seen in both cases, indicating that Woodchuck CD28 and

CTLA-4 were expressed in transfected cells and detectable by corre-

sponding antibodies (Fig. 3).

Detection of Woodchuck CD28 and CTLA-4 on woodchuck

lymphocytes

To detect Woodchuck CD28 and CTLA-4 on woodchuck lympho-

cytes, PHA-activated woodchucks PBMCs were stained with CD28-

and CTLA-4-specific antibodies used previously for indirect

immunofluorescence staining of transfected cells (Fig. 4). Consistent

with the fact that CD28 and CTLA-4 are cell-surface antigens, the

staining of woodchuck PBLs was associated with cytoplasmic

membranes.

Discussion

In this present work, we characterized Woodchuck CD28 and CTLA-

4 by molecular cloning of cDNAs comprising their coding regions.

The comparison of Woodchuck CD28 and CTLA-4 with their mam-

malian homologues showed that woodchuck is genetically more close

to human than to mouse or rat, consistent with the previous study on

Fig. 1. Comparison of the sequences of Woodchuck CD28 and

CTLA-4 cDNAs. Only the putative coding regions of these genes were

considered. Dots resemble conserved nucleotide residues and gaps (–) were

inserted to maximize alignment. Nucleotide positions conserved across CD28

and CTLA-4 of different mammalian species are underlined.

Similarity of Woodchuck CD28 and CTLA-4 to the respective sequences of other

species on the amino-acid level.

CD28 CTLA-4

Human 76% 86%

Monkey 74% 85%

Bovine 76% 80%

Sheep 76% 80%

Pig – 82%

Dog 79% 83%

Mouse 70% 75%

Rat 73% 78%

Rabbit 79% 85%

Chicken 52% –

Sequences of CD28 and CTLA-4 from different species are published in (21,40,44–54).

Table 2

Lu et al : Woodchucks CD28 and CTLA-4

228 Tissue Antigens 2003: 62: 225–232

Fig. 2. Alignment of deduced amino-acid sequence of Woodchuck CD28 (A) and CTLA-4 (B) with homologues from different species. Dots

resemble conserved amino acids, and gaps (–) were inserted to maximize alignment. The sequences of CD28 and CTLA-4 from different species are published

in (44–50).

Lu et al : Woodchucks CD28 and CTLA-4

Tissue Antigens 2003: 62: 225–232 229

woodchuck cytokines (10). The conservation of all known critical

amino-acid residues and motives in Woodchucks CD28 and CTLA-4

suggests that both proteins are functionally equivalent with their

homologues of other species.

The respective proteins were expressed by the cloned cDNAs of

Woodchuck CD28 and CTLA-4 and detection in transfected BHK

cells by immunofluorescence staining with specific antibodies. The

availability of these specific antibodies also allowed us to detect

Woodchuck CD28 and CTLA-4 expressed on cultured T cells by

immunohistochemical staining. This will facilitate studies on the

expression of CD28 and CTLA-4 on woodchuck lymphocytes in the

context of hepadnavirus infection.

Woodchuck is an important animal model to study the hepatitis

B virus infection. One of the major challenges is to understand the

molecular mechanisms leading to virus elimination or virus persis-

tence. This knowledge about the components of the immune system

of woodchuck will help to define the factors that determine the

course of infection. Particularly, the available cDNAs of Wood-

chucks CD28 and CTLA-4 can be used to generate recombinant

chimera proteins with IgG for studies on T-cell activation during

a virus infection in woodchucks. Furthermore, Boyle et al. showed

that a fusion protein of CTLA-4 and IgG leads to an efficient

immune response to the IgG domain, presumably as a result of

the direction of the antigen to antigen-presenting cells by the inter-

action between the B7 and CTLA-4 molecules (43). Thus, it will be

worthy to test recombinant fusion proteins consisting of Wood-

chuck CTLA-4 and viral antigens for prophylactic and therapeutic

vaccinations in the future.

Fig. 3. Immunofluorescence staining of baby hamster kidney cells.

(A) Cells transfected with peCD28 and stained with goat anti-CD28 IgG. (B)

Cells transfected with pcDNA3 and stained with goat anti-CD28 IgG. (C). Cells

transfected with peCTLA4 and stained with goat anti-CTLA-4 IgG. (D). Cells

transfected with pcDNA3 and stained with goat anti-CTLA-4 IgG.

Fig. 4. Immunohistochemical staining of phytohemagglutinin (PHA)-activated woodchuck peripheral blood lymphocytes with antibodies to

CD28 and CTLA-4. (A) Control staining with non-specific goat IgG as primary antibodies. (B) Staining with goat anti-CD28 IgG. (C) Staining with goat anti-

CTLA-4 IgG.

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