Conservation of a CD1 Multigene Family in
the Guinea Pig
This information is current as
of June 16, 2017.
Christopher C. Dascher, Kenji Hiromatsu, Jerome W.
Naylor, Pamela P. Brauer, Kara A. Brown, James R. Storey,
Samuel M. Behar, Ernest S. Kawasaki, Steven A. Porcelli,
Michael B. Brenner and Kenneth P. LeClair6
J Immunol 1999; 163:5478-5488; ;
http://www.jimmunol.org/content/163/10/5478
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Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Conservation of a CD1 Multigene Family in the Guinea Pig1
Christopher C. Dascher,* Kenji Hiromatsu,* Jerome W. Naylor,2† Pamela P. Brauer,3†
Kara A. Brown,4† James R. Storey,‡ Samuel M. Behar,* Ernest S. Kawasaki,5†
Steven A. Porcelli,* Michael B. Brenner,* and Kenneth P. LeClair6†‡
R
ecent structural and functional data have led to the identification of the CD1 family as a third lineage of Agpresenting molecules distinct from those encoded by the
MHC (reviewed in Ref. 1). CD1 proteins are expressed as heterodimeric cell-surface glycoproteins consisting of an ;45-kDa
mature glycosylated H chain that is noncovalently associated with
b2-microglobulin (b2m).7 CD1 H chains are type I integral membrane proteins that consist of three extracellular domains (a1, a2,
and a3) similar in size to those of MHC class I, followed by a
transmembrane segment and short cytoplasmic tail at the carboxyl
terminus. Analysis of protein sequence data demonstrate that the
CD1 family shares approximately equal homology with both MHC
class I and class II. This suggests that CD1 diverged from a common ancestral gene for MHC proteins at a distant point in vertebrate evolution, possibly close to the time that MHC class I and
class II diverged from each other. Consistent with this proposed
*Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115; †Procept, Cambridge, MA;
and ‡Aquila Biopharmaceuticals, Framingham, MA 01702
Received for publication June 25, 1999. Accepted for publication September 7, 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
This work was supported by grants from the American Cancer Society (S.A.P.) and
the National Institutes of Health (C.C.D., S.A.P., and M.B.B.). K.H. is supported by
a Postdoctoral Fellowship Award from the Medical Foundation. K.L. and P.B. were
supported by an Small Business Innovation Research Grant from the National Institutes of Health (AI-40798).
2
Current address: Massachusetts Institute of Technology Genome Center, Cambridge, MA 02139.
3
Current address: Millennium BioTherapeutics, Cambridge, MA 02139.
4
Current address: Ariad Pharmaceuticals, Cambridge, MA 02139.
5
Current address: GSI Lumonics, Watertown, MA 02154.
6
Address correspondence and reprint requests to Dr. Ken LeClair, Aquila Biopharmaceuticals, 175 Crossing Boulevard, Framingham, MA 01702. E-mail address:
[email protected]
Abbreviations used in this paper: b2m, b2-microglobulin; RACE, rapid amplification of cDNA ends; GPI, glycosylphosphatidylinositol.
7
Copyright © 1999 by The American Association of Immunologists
distant evolutionary relationship to the MHC, genetic mapping has
shown the CD1 locus in humans and mice to be unlinked to the
MHC in both species.
Human CD1 represents a small multigene family composed of
five nonpolymorphic members, designated CD1a, -b, -c, -d, and -e.
The five CD1 protein sequences are divided into two groups based
on homology, with group 1 comprised of CD1a, -b, and -c and
group 2 comprised of only CD1d (1, 2). The CD1e protein sequence has an intermediate homology between group 1 and 2 (2).
Group 1 CD1 proteins are expressed at the surface of a wide range
of APCs, including Langerhans cells, dendritic cells, B cells, and
cytokine-activated monocytes (reviewed in Ref. 1). While the tissue distribution and gene sequence data for CD1 proteins have
been known for some time, only recently has the function of CD1
been appreciated. Initial studies established a role for the human
CD1b protein in the recognition of an Ag derived from Mycobacterium tuberculosis by a CD42CD82 T cell line (3). These studies
have been extended by the characterization of additional CD1restricted T cell lines that now include examples belonging to most
of the major phenotypic T cell subsets, including CD41CD82,
CD42CD81, and CD42CD82 TCR-ab1 T cells (3– 6). In addition, gd TCR1 T cells recognizing CD1 proteins have also been
described (3, 7).
The first isolation of an Ag recognized by a CD1b-restricted
human T cell line by Beckman et al. yielded the surprising finding
that the M. tuberculosis Ag presented to these T cells was mycolic
acid, an abundant mycobacterial cell wall lipid (8). A greater appreciation of the structural features of CD1 Ags is now emerging
as the number of defined CD1-presented lipid and glycolipid Ags
increases (9 –12). All of the CD1-presented Ags identified to date
have two hydrophobic lipid tails coupled to a charged or hydrophilic head group (e.g., a carboxylate, simple sugar, or oligosaccharide) (13, 14). Studies of T cell recognition of these Ags have
shown exquisite specificity for the hydrophilic head group structure, but not for the hydrophobic lipid tails (10). This supports the
prediction that the hydrophilic head groups of Ags bound to CD1
0022-1767/99/$02.00
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CD1 is a family of cell-surface molecules capable of presenting microbial lipid Ags to specific T cells. Here we describe the CD1
gene family of the guinea pig (Cavia porcellus). Eight distinct cDNA clones corresponding to CD1 transcripts were isolated from
a guinea pig thymocyte cDNA library and completely sequenced. The guinea pig CD1 proteins predicted by translation of the
cDNAs included four that can be classified as homologues of human CD1b, three that were homologues of human CD1c, and a
single CD1e homologue. These guinea pig CD1 protein sequences contain conserved amino acid residues and hydrophobic domains
within the putative Ag binding pocket. A mAb specific for human CD1b cross-reacted with multiple guinea pig CD1 isoforms, thus
allowing direct analysis of the structure and expression of at least a subset of guinea pig CD1 proteins. Cell-surface expression of
CD1 was detected on cortical thymocytes, dermal dendritic cells in the skin, follicular dendritic cells of lymph nodes, and in the
B cell regions within the lymph nodes and spleen. CD1 proteins were also detected on a subset of PBMCs consistent with expression
on circulating B cells. This distribution of CD1 staining in guinea pig tissues was thus similar to that seen in other mammals. These
data provide the foundation for the development of the guinea pig as an animal model to study the in vivo function of CD1. The
Journal of Immunology, 1999, 163: 5478 –5488.
The Journal of Immunology
Materials and Methods
Animals
Hartley guinea pigs were obtained from Charles River Breeders (Willmington, MA). Strain 2 guinea pigs were obtained from the National Cancer
Institute (Frederick, MD). Animals were housed under specific pathogenfree conditions at the Brigham and Women’s Hospital Animal Facility. All
appropriate animal use protocols were obtained and followed.
Preparation of cells and tissues
Guinea pig whole blood was obtained by cardiac puncture of anesthetized
animals. PBMC were isolated by Ficoll gradient as described previously
(20). Thymus tissue was obtained from 3-wk-old guinea pigs. Other tissues
were obtained from adult animals. Single-cell suspensions of spleen, thymus, and lymph nodes were generated by mincing tissue and passing
through a no. 60 wire mesh screen. Cell suspensions were washed three
times in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% FCS. The guinea pig cell lines used here were obtained from
the American Type Culture Collection (Manassas, VA) and included
104C1 (CRL-1405), JH4 (CCL-158), and GP16 (CCL-242). These cells
were grown in DMEM (11995-065; Life Technologies) with 10% FCS
(HyClone, Logan, UT), 100 U/ml penicillin/streptomycin (15140-122; Life
Technologies), 60 mM nonessential amino acids (1140-050; Life Technologies), and 0.8 mM L-glutamine (25030-081; Life Technologies). Cells
were cultured in a humidified 10% CO2 incubator at 37°C.
Southern blot and hybridization analysis
Genomic DNA was isolated from the livers of Hartley or strain 2 guinea
pigs using standard extraction techniques (21). Mouse and human genomic
DNA were gifts from Drs. Christina Parker and Hamid Band, respectively.
Southern blotting and hybridization were performed as described elsewhere (21). Briefly, genomic DNA was digested with restriction enzymes
and electrophoresed on a 0.7% agarose gel, blotted onto Hybond-N membrane (Amersham Life Sciences, Little Chalfont, U.K.), and cross-linked
by a 3-min exposure to UV light. The blot was prehybridyzed at 42°C for
2 h with prehybridyzation solution (53 SSC, 53 Denharts, 50% formamide, 50 mM HEPES, pH 7.0, 0.5% SDS, and 160 mg/ml salmon sperm
DNA). The probe was generated by PCR with 59 primer (59-AGTGAA
CATGCCTTCCAGGGGCCGACC-39) and 39 primer (59-GGGGTCGAC
GAGGATGATGTCCTGGCC-39) to generate a product containing the
nucleotides encoding the a1, a2, and a3 domains of the human CD1b
cDNA. The CD1b probe was radioactively labeled with [32P]dCTP using
the Rediprime random primer system (Amersham Life Sciences). Southern
blots were hybridized with the CD1b probe overnight at 42°C. Blots were
washed sequentially as described (22) and then exposed to X-Omat AR
film (Eastman Kodak, Rochester, NY).
Cloning and sequencing of guinea pig CD1 genomic fragments
and cDNA clones
Genomic DNA was used as the template for PCR using combinations of
consensus oligonucleotide primers corresponding to the beginning and end
of the CD1 a3-encoding genomic DNA exon. The four forward primers
were 59-CCHGARGCCTGGCTGTCC-39, 59-CCHGARGCCTGGCTTT
CC-39, 59-CCHGARGCTTGGCTGTCC-39, 59-CCANYCCTGGGYCTG
GCC-39; and the four reverse primers were 59-CCTAGACTGCTGTG
BYTCACTC-39, 59-CCTAGACTGCTGTGBYTCACCC-39, 59-CCTAGA
CTGCTGTGBYTCACTT-39, 59-CCTAGACTGCTGTGBYTCACCT-39.
Ambiguity codes for these primers are B 5 C, T, or G; H 5 A, C, or T;
M 5 A or C; N 5 C, A, T, or G; R 5 A or G; Y 5 C or T. Typical PCRs
were performed with 266 nM concentrations of each primer and 1 mg
genomic DNA template in a reaction buffer containing 1.5 mM MgCl2, 50
mM of each dNTP, and 1.25 U Taq polymerase for 30 cycles on a PerkinElmer thermal cycler (Norwalk, CT) with annealing temperatures between
45 and 55°C.
For isolation of CD1 cDNAs, total thymocyte RNA was prepared from
Hartley or strain 2 guinea pig thymus tissue by homogenization and extraction with TRIzol as recommended by the manufacturer (Life Technologies). First-strand cDNA was reverse-transcribed from oligo(dT) or random-primed RNA templates using Superscript II reverse transcriptase (Life
Technologies). Alternatively, mRNA was prepared by oligo(dT) priming
of total RNA isolated from thymus tissue using a FastTrack 2.0 mRNA
isolation kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA libraries
were prepared from thymus RNA template using a Marathon cDNA amplification kit (Clontech Laboratories, Palo Alto, CA). Marathon cDNA
adapter ends containing AP1 and AP2 sequences were ligated to the double-stranded cDNAs to facilitate obtaining full-length CD1 cDNAs using
rapid amplification of cDNA ends (RACE) methodologies. In this process,
PCRs from thymus cDNA template were performed using a gpCD1 genespecific internal primer paired with either the AP1- or the AP2-specific end
primers.
PCR-derived genomic DNA and cDNA insert fragments were ligated
into the pCR2.1 TA cloning vector and used to transform Escherichia coli
strain INVaF9 competent cells (Invitrogen). Miniprep DNA was prepared
using the Wizard DNA purification systems (Promega, Madison, WI).
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proteins are exposed to the aqueous environment and thus accessible for direct recognition by TCRs. Recognition of the CD1-lipid
complex by the TCR-ab has recently been confirmed by TCR
transfection experiments (15).
Substantial insight into the mechanism by which CD1 proteins
can bind and present lipid Ags was recently provided by the elucidation of the crystal structure of the mouse CD1d1 protein (16).
This revealed a remarkable resemblance in the overall three-dimensional structure of CD1 to that of MHC class I, with two antiparallel a1 and a2 helices forming the sides of a putative Ag
binding pocket and a b-pleated sheet forming the floor of the
pocket. However, compared with a typical MHC class I molecule,
the a1 and a2 helices are closer together and more elevated above
the b sheet platform. This creates an Ag binding pocket that is
deeper and larger in volume than the MHC class I groove. Importantly, the CD1 Ag binding groove is lined primarily with nonpolar
or hydrophobic amino acids, making its molecular surface electrostatically neutral with essentially no capacity for hydrogen
bonding. These characteristics are consistent with its potential to
bind hydrophobic lipids rather than peptides.
Whereas humans possess both group 1 (CD1a, -b, -c) and group
2 CD1 (CD1d) proteins, the genomes of muroid rodents (mice and
rats) appear to contain only genes encoding homologues of the
group 2 CD1d protein (17, 18). Studies from our laboratory and
others demonstrate that in vitro-derived CD1-restricted T cell lines
specific for mycobacterial lipid Ags can be restricted by one of the
group 1 CD1 proteins, either CD1a, CD1b, or CD1c (5, 8 –10, 19).
In contrast, no CD1d-restricted T cells have been demonstrated
against bacterially derived Ags, although CD1d has been shown to
present GPI-linked proteins derived from protozoal pathogens
(11). Moreover, while most group 1 CD1-reactive T cells secrete
Th1 cytokines, murine CD1d-reactive T cells have been shown to
rapidly secrete high levels of IL-4 following activation and, therefore, may function more as immunoregulatory T cells than as effectors in the control of invading pathogens (4). Thus it is possible
that group 1 and group 2 CD1 proteins have distinct roles in the
host immune response, either with respect to the effector functions
of the CD1-restricted T cells or with respect to the types of Ags
presented by each group.
Investigation of the role of group 1 CD1 proteins in vivo requires an animal model in which group 1 CD1 proteins are expressed. The mouse, having only group 2 CD1, cannot be used as
a model for the study of group 1 CD1 in vivo. Therefore, characterization of other potential animal models is needed. Here we
report the cloning and analysis of the guinea pig CD1 gene family
and demonstrate the expression of CD1 proteins by cells in a variety of lymphoid and nonlymphoid tissues. Our findings demonstrate that, unlike the muroid rodents, guinea pigs have an extended family of CD1 genes that includes genes encoding clear
evolutionary homologues of the human CD1b, CD1c, and CD1e
proteins. We propose that the guinea pig provides a uniquely relevant small animal model for in vivo studies of the functions of
group 1 CD1 proteins in infectious disease, autoimmunity, and
cancer.
5479
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GUINEA PIG CD1 GENE FAMILY
Larger quantities of DNA were produced using endo-free plasmid maxiprep kits (Qiagen, Valencia, CA).
DNA sequencing and data analyses
Sequencing of DNA inserts was performed enzymatically by the dideoxy
method using the Sequenase version of T7 DNA polymerase (United States
Biochemicals, Cleveland, OH) with 32P- or 35S-labeled deoxynucleotide
incorporation. DNA sequencing reactions were resolved by electrophoresis
on denaturing polyacrylamide gels followed by XAR-5 film (Eastman
Kodak) autoradiography. Alternatively, automated DNA sequencing was
performed on Applied Biosystems model 373 or 377 instruments at the
Molecular Biology Core Facility of the Dana-Farber Cancer Institute (Boston, MA). Nucleotide and protein sequences obtained were compared with
currently available sequences in the GenBank database using the BLAST
program (23, 24). Alignment and dendrogram of CD1 protein sequences
were made using the PILEUP program from the Wisconsin GCG software
package (25).
Transfection of cells with a guinea pig CD1b3 gene
mAbs and FACS
The mAbs used in these studies include CT6 and CT7 (specific for guinea
pig CD8 and CD4, respectively) (Serotec, Raleigh, NC); BCD1b3.1 (specific for human CD1b and cross-reactive with guinea pig CD1 proteins) has
been described previously (26); PCA/188A was used as anti-CD3 (27). P3
was used as the nonspecific mouse IgG1 isotype control Ab (28). FACS
analysis was performed as previously described (29). Briefly, primary Abs
were added to single-cell suspensions at saturating concentrations for 1 h,
washed with staining buffer (PBS containing 2% FCS and 0.01% azide),
and then incubated for 1 h with 30 mg/ml FITC-conjugated donkey antimouse IgG (Jackson Immunologicals, West Grove, PA). After staining,
cells were washed with staining buffer and analyzed with a FACSort flow
cytometer (Becton Dickinson, Mountain View, CA). The forward and side
scatter profiles of the analyzed cells (PBMC, splenocytes, and lymph node
lymphocytes) were used to gate on the lymphocyte subpopulation. Dead
cells were excluded using propidium iodide (Sigma, St. Louis, MO).
Cell-surface labeling and immunoprecipitation
Freshly isolated thymocytes (2 3 107) were cell-surface labeled with
Na125I (DuPont-New England Nuclear, Boston, MA) using lactoperoxidase
and hydrogen peroxide as described previously (30). The cells were solubilized in lysis buffer (50 mM Tris, pH 7.6, 140 mM NaCl (TBS) with 0.5%
Triton X-100, 16 mM iodoacetamide, and 1 mM PMSF) for 1 h. After
centrifugation to remove insoluble debris, the lysates were precleared with
200 ml of a 10% suspension of Staphylococcus aureus Cowen strain I
(Pansorbin, Calbiochem, La Jolla, CA). Precleared lysates containing 106
cell equivalents were immunoprecipitated with ;1 mg of purified
BCD1b3.1 mAb or 1 ml of P3, CT6, or CT7 ascites followed by incubation
with 40 ml of a 10% suspension of protein A-Sepharose (Pharmacia LKB
Biotechnology, Uppsala, Sweden). The immunoprecipitates were washed
five times with TBS containing 0.1% Triton X-100, then eluted with sample buffer and analyzed by SDS-PAGE using a 12% polyacrylamide gel
under reducing conditions as described (31).
Immunohistochemistry
Tissue samples were mounted in OCT compound (Tissue-Tek, Torrance,
CA), frozen in liquid nitrogen, and stored at 280°C. Frozen tissue sections
(5 mm thick) were fixed in acetone for 10 min, air dried, and stained by an
indirect immunoperoxidase method using avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) and 3-amino-9-ethylcarbazole
(Sigma) as the chromogen.
FIGURE 1. Southern blotting analysis of genomic DNA. Genomic
DNA isolated from guinea pig (lanes 1–3), mouse (lanes 4 – 6), and human
(lanes 7–9) was digested with different restriction enzymes as indicated.
The blot was hybridized overnight at 42°C with a radiolabeled probe containing the a1–a3 exons from human CD1b cDNA. The blot was then
washed twice with 63 SSC at room temperature, twice with 63 SSC at
65°C, and again with 63 SSC at room temperature before autoradiography.
The position of DNA markers (kb) are indicated.
Results
Presence of multiple CD1 genes in the guinea pig
The absence of group 1 CD1 proteins (CD1a, -b, and -c) in the
mouse prompted us to investigate alternative small laboratory animal model systems for functional studies of group 1 CD1 proteins.
Given its more distant relationship to muroid rodents (32, 33), we
chose to investigate whether the guinea pig CD1 repertoire included group 1 CD1 homologues. Preliminary experiments to address this question were conducted using Southern blot analysis.
Genomic DNA obtained from an outbred Hartley guinea pig was
analyzed by Southern blot using a 32P-labeled probe generated
from human CD1b cDNA containing the nucleotides encoding the
a1–a3 exons. The a3 exon of CD1 is the most highly conserved
among all known CD1 protein sequences and was included to
maximize cross-species hybridization of the probe. The resulting
autoradiogram revealed a minimum of 11 bands, suggesting that a
relatively large CD1 family exists in the guinea pig (Fig. 1, lanes
1–3). Samples of mouse and human DNA were analyzed simultaneously and exhibited the expected banding pattern as described in
published reports (34, 35). Five bands corresponding to the five
CD1 genes were detected in the lanes containing human genomic
DNA (Fig. 1, lanes 7–9). Two weakly cross-reactive bands, corresponding to the CD1D1 and CD1D2 genes, were observed with
mouse genomic DNA (Fig. 1, lanes 4 – 6). The absence of nonspecific bands in the human and mouse samples indicates that the
hybridization and wash conditions employed were stringent
enough to detect guinea pig CD1 genes, yet excluded other more
distantly related sequences within the genome (e.g., MHC class I
and class II). Subsequent Southern blot analysis of genomic DNA
from another individual Hartley guinea pig and from the inbred
strain 2 guinea pig resulted in an identical pattern of bands to that
shown in Fig. 1 (data not shown). These data suggested that the
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A cDNA for the gpCD1b3 gene was subcloned into the pcDNA3.1 eukaryotic expression vector (Invitrogen) and transfected into recipient cells
using Lipofectamine Plus lipofection reagents and methodologies (Life
Technologies). Briefly, subconfluent target cells in OptiMEM (Life Technologies) were treated with plasmid DNA and Lipofectamine Plus reagent
for 3–5 h. Cells were then rinsed with and cultured in DMEM with 10%
FCS (HyClone) overnight. To establish a stable gpCD1b3-expressing cell
line, transfected cells were transferred into fresh DMEM with 10% FCS
containing 0.5– 0.75 mg/ml G418 (Life Technologies) for drug selection.
Drug-resistant cells were cloned by limiting dilution and sterile sorted by
flow cytometry using the human CD1b-specific mAb BCD1b3.1.
The Journal of Immunology
5481
guinea pig genome contains an extended nonpolymorphic CD1
gene family with ;11–14 genes.
Cloning and sequencing of guinea pig CD1 a3 exon genomic
DNA fragments
Molecular cloning and sequencing of multiple CD1 genes was undertaken to determine the diversity within the guinea pig CD1
family. To clone guinea pig CD1 genes, we took advantage of the
high degree of nucleotide sequence conservation observed among
CD1 genes, especially in the a3 exon. We aligned all of the published a3-encoding nucleotides and were able to identify multiple
consensus sequences at the beginning and end of the a3 exon.
Multiple PCRs were performed with pairs of these consensus, or
slightly degenerate, synthetic primers using liver genomic DNA as
a template to obtain guinea pig CD1 a3 DNA fragments (Fig. 2A).
Sequencing of the individual genomic a3 fragments yielded 13
distinct CD1 a3 nucleotide sequences. The sequences of three of
these fragments revealed the presence of frame shifts, stop codons,
or both within the a3 exon (data not shown). This suggests that the
guinea pig genome contains CD1 genes that are incapable of
encoding functional CD1 protein and are referred to here as pseudogenes. We have submitted these pseudogene sequences to
GenBank (accession numbers AF178942, AF178943, and
AF178944). The cloning of full-length cDNAs for these three CD1
pseudogenes was not performed. In contrast, the nucleotide and
deduced amino acid sequences of the remaining 10 CD1 gene fragments were consistent with intact genes. Determination of the precise nucleotide sequences of these 10 CD1 gene fragments allowed
us to design a strategy to isolate the full-length cDNAs.
Cloning and analysis of cDNAs encoding guinea pig CD1
proteins
It has been shown previously that all of the CD1 proteins are
coexpressed on cortical thymocytes during thymic development
in humans (36). We prepared adapter-ligated, doubled-stranded
cDNAs from guinea pig thymocyte mRNA to serve as template for
the isolation of guinea pig CD1 cDNAs. Using the a3 nucleotide
sequences, we were able to specify gene-specific PCR primers for
the isolation of corresponding full-length CD1 cDNA sequences
using the RACE technique (Fig. 2, B and C). This strategy allowed
us to isolate full-length cDNAs for the 10 guinea pig CD1 genes.
Of these 10 genes, we identified two additional apparent CD1
pseudogenes (see below).
The nucleotide and predicted amino acid sequences of the remaining eight distinct full-length guinea pig CD1 cDNAs were
submitted for homology analysis to other genes and proteins in the
available databases using the BLAST program (24). Results of
these homology analyses allowed us to classify the various guinea
pig CD1 sequences by reference to the human CD1 genes and
proteins. For each CD1 gene classification we received, both the
nucleotide and amino acid homology were in agreement. The
amino acid sequences of the eight full-length guinea pig CD1 proteins were aligned to each other as shown in Fig. 3.
We then investigated the relationship between the guinea pig
CD1 protein sequences and CD1 proteins from other species. Because the CD1 a1 and a2 domains form the putative Ag binding
cleft, the CD1 protein isoforms are most divergent in these domains. Therefore, we focused on the amino acids comprising these
two domains to evaluate the similarity of the guinea pig CD1 proteins to each other and to CD1 proteins from other species. Fig. 4
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FIGURE 2. Cloning strategy for isolation of guinea pig CD1 cDNA. Consensus oligonucleotide primers corresponding to the beginning and end of the
CD1 a3-encoding genomic DNA exon were used to amplify the a3 exons of multiple CD1 isoforms (A). Individual PCR fragments were cloned, sequenced,
and used to generate specific oligonucleotide primers for each unique CD1 gene for use in subsequent RACE reactions. A guinea pig thymocyte cDNA
library was generated and then ligated at both ends with known oligonucleotide adapter primers AP1 and AP2. Specific oligonucleotide primers were
selected for 59- and 39-end-directed RACE PCR (B and C). For the RACE technique, a gpCD1 a3 gene-specific oligonucleotide and a primer (AP1 or
AP2)-specific oligonucleotide pair was used to generate either 59- or 39-end-directed RACE extension products. This process yielded overlapping cDNA
fragments: one extending from the 59-untranslated region to the 39-end of the a3-encoding exon and the other extending from the 59-end of the a3 exon
through the termination codon into the 39-untranslated region. These cDNA fragments were then spliced together if a convenient restriction enzyme site
occurred in the overlapping region (D). Alternatively, an independent PCR was performed using an oligonucleotide primer corresponding to the revealed
cDNA sequence upstream of the initiation codon paired with one from beyond the stop codon to generate full-length PCR products. TM, transmembrane
exon; cyt., cytoplasmic exon; RE, restriction endonuclease site.
5482
GUINEA PIG CD1 GENE FAMILY
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FIGURE 3. Sequence and alignment of guinea pig CD1 proteins. Guinea pig CD1 protein sequences were generated by conceptual translation of cDNA
sequences and aligned with the program PILEUP. The guinea pig isoforms are listed along the left column. Regions of amino acid identity are shaded. A
consensus (con) sequence is displayed along the bottom. The boundaries of the CD1 structural domains are based on human CD1 domain structure and
are indicated below the consensus sequence. Conserved cysteine residues are indicated by ˆ, and termination codons are indicated by p. Dots within the
aligned CD1 sequences are introduced gaps. Dashes in the consensus reflect variable positions where ,60% of residues are identical. The YXXZ endosomal
targeting motif (see Results and Discussion) is underlined in the consensus sequence. The nucleotide and deduced protein sequences of the guinea pig CD1
family were deposited into the GenBank database. The accession numbers are in parentheses: gpCD1b1 (AF145483); gpCD1b2 (AF145484); gpCD1b3
(AF145485); gpCD1b4 (AF145486); gpCD1c1 (AF145487); gpCD1c2 (AF145488); gpCD1c3 (AF145489); gpCD1e (AF145490).
shows a dendrogram that compares the sequence similarity between the guinea pig CD1 proteins and those of several different
animal species. These data revealed the clustering of the individual
guinea pig CD1 isoforms with corresponding CD1 proteins from
human and other species.
The percent amino acid identity between the guinea pig CD1b
isoforms and the human CD1b protein sequence ranged from 57 to
68%, whereas the identity between the guinea pig CD1b group and
guinea pig CD1c group members ranged from 41 to 54%. Therefore, members of specific isoform groups (CD1b or CD1c) from
guinea pigs were more homologous to the human isoform counterpart than to other guinea pig isoform groups. Based on these
criteria, we have assigned the individual guinea pig CD1 genes to
various CD1 isoform groups. Henceforth, we refer to the guinea
pig CD1 proteins as gpCD1 followed by the specific isoform (e.g.,
gpCD1b3). Using these criteria, the guinea pig thymocytes express
The Journal of Immunology
mRNAs encoding four different CD1b proteins (gpCD1b1, -b2,
-b3, -b4), three CD1c proteins (gpCD1c1, -c2, -c3), and one CD1e
(gpCD1e) protein.
Isoforms corresponding to human CD1a and CD1d have not
been isolated with the techniques described here even though several guinea pig tissues, including thymus, skin, and bone marrowderived cell lines, were used as sources for mRNA. Evidence from
Southern blotting analyses suggested that the guinea pig genome
may lack both the CD1a and the CD1d isoforms. Identical blots of
restriction enzyme-digested guinea pig genomic DNA were hybridized separately with probes corresponding to the a1 and a2
exons of the human CD1a, CD1b, or CD1d cDNAs. Hybridization
and washing conditions were adjusted so that multiple cross-hybridizing bands were clearly detected in guinea pig DNA with the
human CD1b probe (data not shown). In contrast, with human
CD1a or CD1d probes of the same specific activity, and identical
hybridization and washing conditions, no hybridizing bands were
detected in the guinea pig DNA lanes (data not shown).
Conservation of structural features in guinea pig CD1 proteins.
The striking homology observed between guinea pig and human
CD1 proteins is reflected in the conservation of several structural
features common to all CD1 members. The overall domain struc-
ture predicted for the guinea pig CD1 proteins is conserved when
compared with that of human CD1 protein sequences. There is an
;18-residue leader peptide at the amino terminus of each protein
sequence followed by the a1, a2, and a3 domains encoding the
extracellular portion of the CD1 protein. As with other species, the
a1 and a2 domains exhibit a greater degree of amino acid sequence variability than the Ig-like a3 domain (Fig. 3). The a3
domain is followed by an ;20-aa transmembrane domain and a
short cytoplasmic tail. Another feature the guinea pig CD1 proteins
share with CD1 proteins of other species is the presence of multiple sites for Asn-linked carbohydrate attachment at canonical
(Asn-X-Ser/Thr) motifs in the a1 and a2, but not in the a3
domains.
The mouse CD1d1 crystal structure indicates two disulfide
bridges within the H chain of the CD1 molecule (16). One bridge
is found between paired cysteine residues within the a2 domain
(Fig. 3, consensus positions 131 and 195) and another between
paired cysteines located within the a3 domain (Fig. 3, consensus
positions 235 and 290). All of the guinea pig CD1 isoforms have
conserved these paired cysteine residues, suggesting an importance
in the formation of CD1 tertiary structure. In fact, the presence of
these cysteine residues is conserved in all known CD1 protein
sequences with the exception of mouse CD1d2, which substitutes
tryptophan for cysteine at position 167 of the a2 domain (2).
The primary amino acid sequences of the guinea pig CD1 a1
and a2 domains contain a high proportion of hydrophobic amino
acids, a common characteristic among CD1 proteins. The positions
of hydrophobic regions within the a1 and a2 domains of the
gpCD1b and gpCD1c groups is similar to that of human CD1b and
CD1c, respectively. Moreover, structural modeling of the guinea
pig CD1 proteins based on the mouse CD1d1 crystal structure
reveals that the putative Ag binding pocket of the guinea pig CD1
proteins is lined primarily by nonpolar residues.
Human CD1b and CD1c have been shown to contain a tyrosinebased or YXXZ motif (where Y 5 tyrosine, X 5 any residue, Z 5
bulky hydrophobic residue) in their cytoplasmic tails that is important for their endosomal targeting. Human CD1a lacks this motif and remains primarily surface localized (M. Sugita and M. B.
Brenner, unpublished observations), whereas CD1b possesses the
YXXZ motif and shows strong steady-state localization to endosomes (37, 38). Interestingly, the gpCD1b3 cytoplasmic domain,
like human CD1a, lacks the YXXZ motif (Fig. 3, consensus position 341–344). In addition, the gpCD1b3 protein sequence, while
still grouping with CD1b, has a lower degree of homology to the
other gpCD1b isoforms than those isoforms have to each other
(Fig. 4). All other guinea pig CD1 proteins, with the exception of
gpCD1e, possess the endosomal targeting motif.
A guinea pig homologue of human CD1e. The gpCD1e sequence
provides the first description of a CD1e homologue in a species
other than human. Both the human and guinea pig CD1e proteins
fall outside the group 1 and group 2 classification scheme (1, 39).
Northern analysis using the a1- and a2-encoding fragment of the
gpCD1e cDNA as a probe revealed high levels of transcript
expression in the guinea pig thymus (data not shown). The human CD1E gene is also transcribed and can be detected by Northern analysis, but no expression of a protein product has been described (39).
Two unusual features were observed in the guinea pig CD1e
cDNA. First, compared with all of the other guinea pig CD1
cDNAs, the gpCD1e cDNA has an additional 33 nucleotides encoding 11 amino acids precisely at the leader exon-a1 exon junction (Fig. 3, consensus positions 19–29). Second, the gpCD1e protein has an extended cytoplasmic tail that lacks the YXXZ
endosomal targeting motif. Both of these features are conserved
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FIGURE 4. Dendrogram of CD1 protein sequences. Multiple protein
sequence alignment of the CD1 a1 and a2 domains only was performed
with the program PILEUP, and a dendrogram representation of these data
was generated. Horizontal distance along the tree is proportional to the
degree of divergence. The guinea pig CD1 protein sequences described in
Fig. 3 are indicated by asterisks. The division of the CD1 protein sequences
into group 1 and group 2 is indicated. Other CD1 protein sequences were
obtained from GenBank and the species are indicated. The non-guinea pig
CD1 proteins and corresponding GenBank accession numbers are as follow: muCD1d1 (mouse CD1d1; I49581 (17)); muCD1d2 (mouse CD1d2;
P11610 (17)); ratcd1d (rat CD1d; I56235 (18)); huCD1d (human CD1d;
P15813 (2)); CtRb1 (rabbit CD1d; B45887 (22)); scd1d (sheep Cd1d;
CAA07200 (45)); scd1a25 (sheep CD1b; CAA85359 (41)); scd1b42
(sheep CD1b; S47246 (41)); huCD1b (human CD1b; P29061 (55)); hucd1c
(human CD1c; P29017 (55)); hucd1a (human CD1a; P06126 (55)); hucd1e
(human CD1e; P15812 (2)).
5483
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GUINEA PIG CD1 GENE FAMILY
A mAb to human CD1b cross-reacts with guinea pig CD1
Given the apparent conservation of CD1 among mammals, we
tested mAbs specific for the CD1 of other species for cross-reactivity to guinea pig CD1. A panel of CD1 reactive mAbs was
assembled and screened by FACS analysis using guinea pig thymocytes as target cells. These cells were chosen because cortical
thymocytes in humans express high levels of all CD1 proteins.
Surprisingly, a significant number of anti-human CD1 Abs were
positive in this preliminary screen (data not shown). One of these
Abs, BCD1b3.1, an anti-human CD1b-specific mAb, was chosen
for more extensive analysis.
To confirm that the Ag recognized by the BCD1b3.1 mAb on
guinea pig cells was in fact CD1, we characterized the anti-human
CD1b cross-reacting Ag by immunoprecipitation of surface-iodinated guinea pig thymocytes. Immunoprecipitations were then
subjected to SDS-PAGE and autoradiography. Radiolabeled species of 45 and 14 kDa were specifically visualized (Fig. 5, lane 4).
These sizes are consistent with the relative mobilities of a glycoslyated CD1 H chain and the b2m L chain, respectively. To further
confirm the identity of the cross-reacting Ag as a CD1 gene product, we expressed the gpCD1b3 cDNA sequence in the guinea pig
104C1 cell line and analyzed the surface expression by FACS with
mAb BCD1b3.1 (Fig. 6). Strong staining of the transfectant was
observed, confirming that the BCD1b3.1 mAb recognized at least
one of the guinea pig CD1 isoforms when expressed on the cell
surface.
Guinea pig thymocytes showed high staining levels with
BCD1b3.1 when used in FACS (Fig. 7A). The profile indicated
that about 90% of thymocytes were highly positive. The pattern
and intensity was similar to that seen when human thymocytes
were stained with this Ab (data not shown). Single-cell suspensions were generated from normal spleen and lymph nodes and
then subjected to flow cytometry after staining with the BCD1b3.1
Ab as described above. A representative experiment using
BCD1b3.1 demonstrated a subpopulation of CD1-positive cells in
the lymphocyte population of lymph node and spleen (Fig. 7, C
and D). A subpopulation of lymphocytes gated from whole PBMC
(11.8%) also stained positively with the Ab (Fig. 7B). These data
indicated the presence of CD1 molecules cross-reactive with human CD1b on lymphocytes in the blood and lymphoid organs of
guinea pigs.
We also investigated the presence of CD1 on mature guinea pig
T cells. Previous data has shown that CD1 is present only on im-
FIGURE 5. Expression of a CD1 heterodimer on guinea pig thymocytes. Guinea pig thymocytes were cell-surface iodinated and lysed with
0.5% Triton X-100. Immunoprecipitation was performed with mAbs P3
(IgG1 isotype control), CT7 (guinea pig CD4), CT6 (guinea pig CD8), and
BCD1b3.1 (human CD1b). Immunoprecipitated proteins were resolved on
an SDS-PAGE gel under reducing conditions. The bracket indicates the
putative guinea pig CD1 H chain and the bottom arrow is consistent with
the m.w. of the b2m L chain (right).
mature T cell precursors in the thymic cortex, but not on more
mature medullary thymocytes or circulating T cells. A guinea pig
T cell line propagated in vitro by repeated stimulation with PHA
and recombinant human IL-2 revealed no staining with BCD1b3.1
(data not shown). This was consistent with the observation in humans that mature T cells lack expression of CD1a, -b, or -c. In
addition, various guinea pig cell lines of nonhemopoietic origin
including GP16, JH4, and the strain 2 guinea pig tumor line 104C1
were also unreactive with this Ab (Fig. 6 and data not shown).
Expression of CD1 proteins in normal guinea pig tissues
The studies described above showed that the mAb BCD1b3.1 recognized at least one guinea pig CD1 isoform, indicating that this
would be a useful reagent for initial characterization of CD1 protein expression in normal guinea pig tissues. Immunoperoxidase
staining of frozen sections of guinea pig thymus with mAb
BCD1b3.1 revealed dense staining of cells in the cortical region
consistent with CD1 expression on immature thymocytes (Fig.
8A). The spleen was also examined for CD1 expression. Positively
stained cells in the white pulp of the spleen were observed with the
BCD1b3.1 mAb (Fig. 8B). To identify the stained cells, two-color
staining was conducted on spleen sections using the CD3-specific
Ab PCA/188A together with BCD1b3.1. Spleen sections simultaneously stained with these two Abs demonstrated that CD31 T
cells and CD1-positive lymphoid cells represented closely associated but distinct populations in the white pulp of the spleen (data
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between the predicted human and guinea pig CD1e protein
sequences.
Identification of several CD1 pseudogenes in the guinea pig genome. Our initial cloning of the guinea pig a3 exon identified
three genomic DNA fragments of CD1 genes that had frame-shift
mutations. In addition to these three pseudogenes, a CD1b-like
cDNA was cloned that was also defective. We have designated this
pseudogene gpCD1Bc1 and deposited the nucleotide sequence
into the GenBank database (accession number AF147503). The
DNA sequence of this cDNA revealed a single nucleotide deletion
at a position ;30 bases into the a2 domain exon resulting in a
frame shift. This deletion was also seen in genomic DNA from
both Hartley and strain 2 guinea pigs and, therefore, was not
merely a strain-specific polymorphism. The second pseudogene
contained a CD1c-related sequence from the middle of the a1
domain through to the stop codon, but lacked a corresponding
CD1-like 59 end. We have designated this pseudogene as
gpCD1Cc1 and have also deposited it into GenBank (accession
number AF159261). These results indicate that the guinea pig genome contains at least five CD1 pseudogenes.
The Journal of Immunology
5485
not shown). This staining pattern was consistent with CD1 being
present in B cell follicles adjacent to the T cells in periarteriolar
lymphoid sheaths. In addition to the spleen, staining within lymph
nodes with the BCD1b3.1 mAb was also consistent with CD1 expression on lymphocyte corona B cells of secondary lymphoid
follicles, while staining of the associated germinal centers was not
observed (Fig. 8E). To confirm the identity of the CD11 cells in
these tissues as B cells, spleen and lymph node serial sections were
stained with anti-guinea pig IgG and BCD1b3.1. This revealed
colocalization of CD1 and IgG on the same cell population, further
supporting the expression of CD1 on B cells in the guinea pig (data
not shown).
The skin was also analyzed for expression of CD1. The elongated morphology and dermal location of stained cells suggested
CD1 staining of dermal dendritic cells in the guinea pig skin (Fig.
8, C and D). Serial sections of skin were also stained with the
anti-guinea pig MHC class II Ab CI13.1 and exhibited a pattern of
staining similar to sections stained with BCD1b3.1, although the
frequency of MHC class II1 to CD11 cells in the dermis was ;3:1
(data not shown). These results indicated the presence of dermal
dendritic cells in the skin, a subpopulation of which were CD11.
No staining of Langerhans cells was observed in the epidermis
with BCD1b3.1. We also observed staining of cells with dendritic
morphology in the paracortex of the lymph node consistent with
expression of CD1 on interfollicular dendritic cells (Fig. 8F).
These cells exhibit extended processes that penetrate between the
surrounding cells. In addition, staining of lung tissue revealed sporadic clusters of cells exhibiting lymphoid morphology consistent
with bronchiolar-associated lymphoid tissue (data not shown). We
have not detected CD1 in the liver or intestine of guinea pigs with
this Ab. These preliminary results show that CD1 proteins are
constitutively expressed on cells of hemopoietic lineages in a pattern similar to that described for several human CD1 proteins.
Discussion
We have identified eight distinct full-length CD1 genes in the
guinea pig. Based on comparison with the human CD1 isoforms,
the eight predicted guinea pig CD1 proteins encoded by these
FIGURE 7. Detection of CD1 expression on guinea pig lymphocytes by
flow cytometry. Thymocytes (A), PBMC (B), lymph node lymphocytes
(C), and splenocytes (D) were stained with control mAb P3 (broken line)
or mAb BCD1b3.1 (solid line) and detected with a FITC-conjugated antimouse secondary antiserum. Samples were then analyzed by FACS. The
histograms shown represent the gated lymphocyte population.
genes include four CD1b-like, three CD1c-like, and one CD1e-like
proteins. To date, this is the largest number of complete CD1 sequences identified from a single animal species. The predicted
guinea pig CD1 protein sequences demonstrate a high degree of
homology with CD1 protein sequences from other animal species,
indicative of the highly conserved nature of the CD1 gene family.
CD1 genes and proteins have been described in a variety of
mammalian species including human (2, 34), mouse (17), rat (18),
rabbit (22), cow (40), sheep (41), pig (42), cat (43), and dog (44).
Nevertheless, there is significant heterogeneity with respect to the
complement of CD1 family members that have been preserved
and, in some cases, expanded during the evolution of different
mammalian species. The multiple isoforms of the CD1B and
CD1C genes in the guinea pig suggest that relatively recent gene
duplication events have occurred to create an extended family of
CD1 genes in this species. This process has generated at least five
CD1B-like genes in the guinea pig, four of which possess fulllength coding sequences, with the fifth being a pseudogene. A
similar phenomenon is seen in sheep (Ovis aries), which has at
least four genes encoding CD1b-like proteins with only two of
these being full-length CD1-coding sequences (41). In addition,
sheep possess at least one homologue of the group 2 CD1d isoform
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FIGURE 6. Recognition of the gpCD1b3 isoform by the mAb
BCD1b3.1. The guinea pig cell line 104C1 was transfected with either
pcDNA3.1 vector (104C1.mock; A) or a pcDNA3 construct expressing the
gpCD1b3 cDNA (104C1.b3; B). The cell lines were stained with isotype
control mAb P3 (broken line) or mAb BCD1b3.1 (solid line) and detected
with an anti-mouse FITC-conjugated secondary antiserum. Samples were
then analyzed by FACS.
5486
GUINEA PIG CD1 GENE FAMILY
(45). Thus, both sheep and guinea pigs have undergone more extensive amplification of specific CD1 isoforms than primates. The
purpose of these multiple versions of specific CD1 isoforms is
unknown, but suggests an ongoing important role and evolutionary
pressure to expand the opportunities for CD1 based immune
responses.
Mice, in contrast to guinea pigs, lack group 1 CD1 homologues
and have only two highly homologous group 2 CD1 genes
(CD1D1 and CD1D2). The rat, a close relative of mice, appears to
have only a single copy of the CD1D gene (46). Members of the
order Lagamorpha (rabbits and hares) and Rodentia (mice, rats and
guinea pigs) are closely related and together form the cohort Glires
(47). The monophyly of rodents and lagamorphs implies a common ancestor. Because both group 1 and group 2 CD1 genes have
been described in rabbits (22), the most likely explanation for the
lack of group 1 CD1 in muroid rodents is that these genes were
deleted during evolution. Our finding of group 1 CD1 homologues
in guinea pigs further suggests that the loss of group 1 from muroid
rodents occurred after the division of the rodent order into two
major suborders: Sciurognathi containing rats and mice and Hystricognathi containing guinea pigs. This split is estimated to have
occurred ;55 million year ago during the early Eocene (R. Honeycutt, unpublished observations).
Recent molecular phylogenetic data suggest that the guinea pig
is more distantly related to muroid rodents than was previously
indicated by morphological criteria. It has been postulated that
guinea pigs and their relatives (Hystricognathi) may even represent
a distinct order separate from the order Rodentia (32, 33). While
this classification is not generally accepted (48), the evolutionary
divergence between guinea pigs and other members of the order
Rodentia may contribute to the differences we have described between the CD1 gene families of muroid rodents and guinea pigs.
More likely, these differences reflect diverse evolutionary pressures between closely related species that may be acting to remove
or duplicate existing CD1 genes. Given the ability of CD1 proteins
to function as Ag-presenting molecules, environmental influences,
such as exposure to different pathogens, may have exerted selective pressures to alter the number or expression of different CD1
genes in different animal species.
One hypothesis for the presence of multiple isoforms of CD1 in
a given species is to facilitate sampling of Ags from different compartments within the APC. The human CD1b protein, for example,
has been shown to traffic to late endosomes, colocalizing with
MHC class II and HLA-DM molecules (37). This was shown to be
dependent upon the presence of an intact YXXZ endosomal targeting motif (37, 38). In contrast, human CD1a protein lacks the
YXXZ targeting motif and shows substantially lower steady-state
accumulation in endosomes, suggesting that human CD1a and
CD1b mediate distinct pathways for Ag presentation (M. Sugita
and M. B. Brenner, unpublished observations). We have not identified a guinea pig homologue of human CD1a. However, the
gpCD1b3 protein also lacks the critical YXXZ endosomal targeting motif at the carboxyl terminus of the protein. Based on previous data, this suggests that the gpCD1b3 protein may have a trafficking pattern similar to human CD1a. With the exception of
gpCD1b3 and gpCD1e, all of the guinea pig CD1 proteins possess
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FIGURE 8. Expression of CD1 in guinea pig tissues. Frozen sections of thymus (A), spleen (B), trunk skin (C and D), and lymph node (E and F) were
stained with the mAb BCD1b3.1. Stained sections were visualized with an anti-mouse avidin-biotin-peroxidase complex. Positive staining appears red in
these sections. A shows staining of the cortex in a thymic lobule from a 3-wk-old guinea pig (3100). B shows a cross section of spleen with staining in
the white pulp (3100). C shows cells of dendritic morphology staining in the dermal layer of the trunk skin (3100). D shows the surface staining of CD1
on cells with dendritic morphology in the trunk skin dermis (3600). E shows an adult inguinal lymph node with staining of the lymphocyte corona of two
secondary B cell follicles and negative staining in the associated germinal centers (3100). F is centered on the paracortex of the lymph node and shows
staining of numerous cells with dendritic morphology. The edge of a secondary B cell follicle evident in the upper right of F (3200). All tissues were
counterstained with hematoxylin except A.
The Journal of Immunology
B cells may be to present lipid or glycolipid Ags to T cells for the
purpose of eliciting T cell help for the production of Abs to glycolipid Ags (11, 50).
Previous data have shown that the human dendritic cells found
in lymphoid and nonlymphoid tissues express high levels of the
group 1 CD1 isoforms. Immunohistochemistry of guinea pig
lymph nodes also revealed the presence of CD1 on cells with dendritic morphology in the paracortex, which is consistent with interfollicular dendritic cells found in various lymphoid tissues (Fig.
8F). Dendritic cells in the dermal layer of the skin were also observed in guinea pig (Fig. 8, C and D). Dermal dendritic cells in
human skin express CD1a and CD1c with a subpopulation of dermal dendritic cells also expressing CD1b (51). The CD1a isoform
is highly expressed on epidermal Langerhans cells in human skin.
So far, we have failed to identify CD1 staining of epidermal Langerhans cells in guinea pigs, although we have observed these cells
in serial sections using an anti-MHC class II mAb. It is well established that dendritic cells are potent APCs and play a crucial
role in priming specific T cell responses (52). Therefore, the expression of CD1 on interfollicular dendritic cells and dermal dendritic cells in guinea pigs increases the plausibility that these cells
may be involved in the generation of CD1-restricted T cell
responses.
The recent appreciation of the Ag-presenting capacity of CD1
now requires assessment of these molecules in the larger framework of the immune response to infection and other disease processes. Human T cells that respond to CD1-presented lipid and
glycolipid Ags secrete high levels of IFN-g (5), lyse infected cells,
and also have bacteriocidal effects on M. tuberculosis localized
within macrophages (53). These data support a possible role for
CD1-restricted T cells in control or clearance of mycobacterial
infections such as tuberculosis or leprosy (54). However, direct in
vivo proof of a significant role for the group 1 CD1 proteins in host
response to infection has yet to be obtained. Our demonstration
that guinea pigs, like humans, have clearly preserved and expanded the group 1 subset of CD1 Ag-presenting molecules suggests that these proteins may confer an important evolutionary advantage in this species that may be relevant to their role in the
human immune system. The findings presented here thus indicate
that the guinea pig will be useful as a small animal model for
examining the role of group 1 CD1 in tuberculosis and other infections relevant to human disease.
Acknowledgments
We thank Dr. Rodney Honeycutt for valuable comments on rodent evolution, Dr. David McMurray for discussions about the guinea pig animal
model, Dr. Masahiko Sugita for sharing unpublished data, and Robert Carpenter for support of this project. We also thank Drs. Cheryl Murphy,
Charlotte Kensil, and Gerald Beltz for critical review of the manuscript.
References
1. Porcelli, S. A. 1995. The CD1 family: a third lineage of antigen-presenting molecules. Adv. Immunol. 59:1.
2. Calabi, F., J. M. Jarvis, L. Martin, and C. Milstein. 1989. Two classes of CD1
genes. Eur. J. Immunol. 19:285.
3. Porcelli, S., M. B. Brenner, J. L. Greenstein, S. P. Balk, C. Terhorst, and
P. A. Bleicher. 1989. Recognition of cluster of differentiation 1 antigens by human CD42CD82 cytolytic T lymphocytes. Nature 341:447.
4. Bendelac, A., R. D. Hunziker, and O. Lantz. 1996. Increased interleukin 4 and
immunoglobulin E production in transgenic mice overexpressing NK1 T cells.
J. Exp. Med. 184:1285.
5. Rosat, J. P., E. P. Grant, E. M. Beckman, C. C. Dascher, P. A. Sieling,
D. Frederique, R. L. Modlin, S. A. Porcelli, S. T. Furlong, and M. B. Brenner.
1999. CD1-restricted microbial lipid antigen-specific recognition found in the
CD81 ab T cell pool. J. Immunol. 162:366.
6. Porcelli, S., C. T. Morita, and M. B. Brenner. 1992. CD1b restricts the response
of human CD4282 T lymphocytes to a microbial antigen. Nature 360:593.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
a YXXZ motif at the carboxyl terminus. Differences in the subcellular localization of human CD1a and CD1b isoforms potentially allows Ags in different subcellular compartments to be sampled by the APC and subsequently presented to T cells. This may
be a particularly important mechanism for the presentation of Ags
following infection by intracellular pathogens, such as M. tuberculosis, that are capable of parasitizing macrophages. The presence
of multiple CD1b and CD1c isoforms in the guinea pig provides a
unique opportunity to examine the fine specificity of CD1 Ag presentation and may provide additional insight into the specific function of the individual isoforms.
It is currently unclear whether the guinea pig harbors a CD1d
homologue. Although the available data on CD1 in other species
suggests that this member of the family is frequently conserved,
our methods have so far failed to demonstrate CD1d in the guinea
pig. One possible explanation for this is that a putative gpCD1d
sequence may significantly differ from the degenerate primers used
in the initial amplification of the a3 exon from genomic DNA.
This may also explain the absence of cross-hybridizing bands in
Southern blot analyses of guinea pig genomic DNA when high
stringency wash conditions were used with a probe containing the
a1 and a2 exons of human CD1d cDNA. In contrast, identical
Southern blots hybridized with a human CD1b a1 and a2 exon
probe in the same experiment showed multiple cross-hybridizing
bands. Thus, genes encoding guinea pig homologues of CD1d may
be significantly divergent from the human CD1d probes used here,
or these genes may simply be absent in the guinea pig.
Characterization of the expression of guinea pig CD1 proteins
revealed many features similar to CD1 expression in humans. The
apparent molecular mass of the precipitated CD1 protein was almost identical with that of the glycosylated human CD1 protein
and revealed a similar association with b2m (Fig. 5). CD1 expression was detected by FACS analysis of normal guinea pig PBMC,
thymocytes, splenocytes, and lymph node cells (Fig. 7). In addition, FACS analysis of a guinea pig CD1 transfectant demonstrated
that the BCD1b3.1 mAb cross-reacted with the gpCD1b3 gene
product (Fig. 6). The expression of CD1 proteins detected by immunohistochemical staining using the BCD1b3.1 mAb revealed
high levels of CD1 on thymocytes (Figs. 5A and 8A). Cortical
thymocytes, which are predominantly immature T cells, express
high levels of CD1 protein during thymic development (36). CD1
protein expression appeared to be lost following maturation of
guinea pig T cells, as evidenced by the absence of CD1 staining on
medullary thymocytes (Fig. 8A). In addition, the CD31 T cell regions in the spleen were negative for CD1 staining, and T cells
propagated in vitro also lacked expression of CD1. More recent
data now demonstrate that, in addition to gpCD1b3, the BCD1b3.1
mAb also reacts with the gpCDb2, -b4, and -c3 isoforms (our
unpublished observations). Because of the lack of monospecific
mAb reagents, we cannot identify the specific guinea pig CD1
isoforms expressed on a particular cell type within tissues.
The staining of CD11CD32IgG1 cells with lymphoid morphology closely associated with periarteriolar lymphoid sheaths is consistent with CD1 expression on B cells in the guinea pig (Fig. 8, B
and E). It has been shown previously that B cells derived from
human spleen and PBMC express the CD1c protein (49). In addition to the spleen, strong staining of secondary lymphoid follicles
in guinea pig lymph nodes is also consistent with CD1 expression
on B cells (Fig. 8E). We have also detected a subpopulation of
CD11 cells in the PBMC of guinea pigs, which, as indicated by
two-color FACS using Abs specific for CD1 and IgG, are most
likely circulating B cells (Fig. 7B and unpublished observations).
These data indicate that B cells are a major population of CD1bearing cells in the guinea pig. One possible function of CD1 on
5487
5488
32. Graur, D., W. A. Hide, and W. H. Li. 1991. Is the guinea-pig a rodent? Nature
351:649.
33. D’Erchia, A. M., C. Gissi, G. Pesole, C. Saccone, and U. Arnason. 1996. The
guinea-pig is not a rodent. Nature 381:597.
34. Martin, L. H., F. Calabi, and C. Milstein. 1986. Isolation of CD1 genes: a family
of major histocompatibility complex-related differentiation antigens. Proc. Natl.
Acad. Sci. USA 83:9154.
35. Moseley, W. S., M. L. Watson, S. F. Kingsmore, and M. F. Seldin. 1989. CD1
defines conserved linkage group border between human chromosomes 1 and
mouse chromosomes 1 and 3. Immunogenetics 30:378.
36. Gambon, F., M. Kreisler, and F. Diaz-Espada. 1988. Correlated expression of
surface antigens in human thymocytes: evidence of class I HLA modulation in
thymic maturation. Eur. J. Immunol. 18:153.
37. Sugita, M., R. M. Jackman, E. van Donselaar, S. M. Behar, R. A. Rogers,
P. J. Peters, M. B. Brenner, and S. A. Porcelli. 1996. Cytoplasmic tail-dependent
localization of CD1b antigen-presenting molecules to MIICs. Science 273:349.
38. Jackman, R. M., S. Stenger, A. Lee, D. B. Moody, R. A. Rogers, K. R. Niazi,
M. Sugita, R. L. Modlin, P. J. Peters, and S. A. Porcelli. 1998. The tyrosinecontaining cytoplasmic tail of CD1b is essential for its efficient presentation of
bacterial lipid antigens. Immunity 8:341.
39. Calabi, F., C. A. G. Bilsland, C.-Y. Yu, A. Bradbury, K. T. Belt, L. H. Martin,
and C. Milstein. 1989. Recent progress in the molecular study of CD1. In Leukocyte Typing IV. W. Knapp, ed. Oxford University Press, Oxford, p. 254.
40. MacHugh, N. D., A. Bensaid, W. C. Davis, C. J. Howard, K. R. Parsons, B. Jones,
and A. Kaushal. 1988. Characterization of a bovine thymic differentiation antigen
analogous to CD1 in the human. Scand. J. Immunol. 27:541.
41. Ferguson, E. D., B. M. Dutia, W. R. Hein, and J. Hopkins. 1996. The sheep CD1
gene family contains at least four CD1B homologues. Immunogenetics 44:86.
42. Chun, T., K. Wang, F. A. Zuckermann, and H. R. Gaskins. 1999. Molecular
cloning and characterization of a novel CD1 gene from the pig. J. Immunol.
162:6562.
43. Woo, J. C., and P. F. Moore. 1997. A feline homologue of CD1 is defined using
a feline-specific monoclonal antibody. Tissue Antigens 49:244.
44. Moore, P. F., M. D. Schrenzel, V. K. Affolter, T. Olivry, and D. Naydan. 1996.
Canine cutaneous histiocytoma is an epidermotropic Langerhans cell histiocytosis that expresses CD1 and specific b2-integrin molecules. Am. J. Pathol. 148:
1699.
45. Rhind, S. M., J. Hopkins, and B. M. Dutia. 1999. Amino-terminal sequencing of
sheep CD1 antigens and identification of a sheep CD1D gene. Immunogenetics
49:225.
46. Ichimiya, S., A. Matsuura, S. Takayama, and K. Kikuchi. 1993. Molecular cloning of a cDNA encoding the rat homologue of CD1. Transplant. Proc. 25:2773.
47. Honeycutt, R. L., and R. M. Adkins. 1993. Higher level systematics of eutherian
mammals: an assessment of molecular characters and phylogenetic hypotheses.
Annu. Rev. Ecol. Syst. 24:279.
48. Nedbal, M. A., R. L. Honeycutt, and D. A. Schlitter. 1996. Higher-level systematics of rodents (Mammalia, Rodentia): evidence from the mitochondrial 12S
rRNA gene. J. Mammal. Evol. 3:201.
49. Small, T. N., R. W. Knowles, C. Keever, N. A. Kernan, N. Collins, R. J. O’Reilly,
B. Dupont, and N. Flomenberg. 1987. M241 (CD1) expression on B lymphocytes. J. Immunol. 138:2864.
50. Fairhurst, R. M., C. X. Wang, P. A. Sieling, R. L. Modlin, and J. Braun. 1998.
CD1-restricted T cells and resistance to polysaccharide-encapsulated bacteria.
Immunol. Today 19:257.
51. Furue, M., M. Nindl, K. Kawabe, K. Nakamura, Y. Ishibashi, and K. Sagawa.
1992. Epitope mapping of CD1a, CD1b, and CD1c antigens in human skin:
differential localization on Langerhans cells, keratinocytes, and basement membrane zone. J. Invest. Dermatol. 99:23S.
52. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity.
Annu. Rev. Immunol. 9:271.
53. Stenger, S., R. J. Mazzaccaro, K. Uyemura, S. Cho, P. F. Barnes, J. P. Rosat,
A. Sette, M. B. Brenner, S. A. Porcelli, B. R. Bloom, and R. L. Modlin. 1997.
Differential effects of cytolytic T cell subsets on intracellular infection. Science
276:1684.
54. Sieling, P. A., D. Jullien, M. Dahlem, T. F. Tedder, T. H. Rea, R. L. Modlin, and
S. A. Porcelli. 1999. CD1 expression by dendritic cells in human leprosy lesions:
correlation with effective host immunity. J. Immunol. 162:1851.
55. Martin, L. H., F. Calabi, F. A. Lefebvre, C. A. Bilsland, and C. Milstein. 1987.
Structure and expression of the human thymocyte antigens CD1a, CD1b, and
CD1c. Proc. Natl. Acad. Sci. USA 84:9189.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
7. Faure, F., S. Jitsukawa, C. Miossec, and T. Hercend. 1990. CD1c as a target
recognition structure for human T lymphocytes: analysis with peripheral blood
g/d cells. Eur. J. Immunol. 20:703.
8. Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, and
M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted ab1 T
cells. Nature 372:691.
9. Sieling, P. A., D. Chatterjee, S. A. Porcelli, T. I. Prigozy, R. J. Mazzaccaro,
T. Soriano, B. R. Bloom, M. B. Brenner, M. Kronenberg, P. J. Brennan, and et
al. 1995. CD1-restricted T cell recognition of microbial lipoglycan antigens. Science 269:227.
10. Moody, D. B., B. B. Reinhold, M. R. Guy, E. M. Beckman, D. E. Frederique,
S. T. Furlong, S. Ye, V. N. Reinhold, P. A. Sieling, R. L. Modlin, G. S. Besra,
and S. A. Porcelli. 1997. Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells. Science 278:283.
11. Schofield, L., M. J. McConville, D. Hansen, A. S. Campbell, B. Fraser-Reid,
M. J. Grusby, and S. D. Tachado. 1999. CD1d-restricted immunoglobulin G
formation to GPI-anchored antigens mediated by NKT cells. Science 283:225.
12. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno,
R. Nakagawa, H. Sato, E. Kondo, H. Koseki, and M. Taniguchi. 1997. CD1drestricted and TCR-mediated activation of va14 NKT cells by glycosylceramides. Science 278:1626.
13. Porcelli, S. A., and M. B. Brenner. 1997. Antigen presentation: mixing oil and
water. Curr. Biol. 7:R508.
14. Porcelli, S. A., B. W. Segelke, M. Sugita, I. A. Wilson, and M. B. Brenner. 1998.
The CD1 family of lipid antigen-presenting molecules. Immunol. Today 19:362.
15. Grant, E. P., M. Degano, J. P. Rosat, S. Stenger, R. L. Modlin, I. A. Wilson,
S. A. Porcelli, and M. B. Brenner. 1999. Molecular recognition of lipid antigens
by T cell receptors. J. Exp. Med. 189:195.
16. Zeng, Z., A. R. Castano, B. W. Segelke, E. A. Stura, P. A. Peterson, and
I. A. Wilson. 1997. Crystal structure of mouse CD1: an MHC-like fold with a
large hydrophobic binding groove. Science 277:339.
17. Bradbury, A., K. T. Belt, T. M. Neri, C. Milstein, and F. Calabi. 1988. Mouse
CD1 is distinct from and co-exists with TL in the same thymus. EMBO J. 7:3081.
18. Ichimiya, S., K. Kikuchi, and A. Matsuura. 1994. Structural analysis of the rat
homologue of CD1: evidence for evolutionary conservation of the CD1D class
and widespread transcription by rat cells. J. Immunol. 153:1112.
19. Beckman, E. M., A. Melian, S. M. Behar, P. A. Sieling, D. Chatterjee,
S. T. Furlong, R. Matsumoto, J. P. Rosat, R. L. Modlin, and S. A. Porcelli. 1996.
CD1c restricts responses of mycobacteria-specific T cells: evidence for antigen
presentation by a second member of the human CD1 family. J. Immunol. 157:
2795.
20. Pollack, V. A., J. S. Brandhorst, and M. G. Hanna, Jr. 1981. Separation of guinea
pig peripheral blood lymphocytes by discontinuous density gradient centrifugation using Ficoll-metrizoate. J. Immunol. Methods 41:29.
21. Ansubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman,
J. A. Smith, and K. Struhl. 1987. Current Protocols in Molecular Biology. John
Wiley and Sons, New York.
22. Calabi, F., K. T. Belt, C. Y. Yu, A. Bradbury, W. J. Mandy, and C. Milstein.
1989. The rabbit CD1 and the evolutionary conservation of the CD1 gene family.
Immunogenetics 30:370.
23. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic
local alignment search tool. J. Mol. Biol. 215:403.
24. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and
D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389.
25. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387.
26. Behar, S. M., S. A. Porcelli, E. M. Beckman, and M. B. Brenner. 1995. A pathway of costimulation that prevents anergy in CD282 T cells: B7-independent
costimulation of CD1-restricted T cells. J. Exp. Med. 182:2007.
27. Jones, M., J. L. Cordell, A. D. Beyers, A. G. Tse, and D. Y. Mason. 1993.
Detection of T and B cells in many animal species using cross-reactive antipeptide antibodies. J. Immunol. 150:5429.
28. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 256:495.
29. Parker, C. M., V. Groh, H. Band, S. A. Porcelli, C. Morita, M. Fabbi, D. Glass,
J. L. Strominger, and M. B. Brenner. 1990. Evidence for extrathymic changes in
the T cell receptor g/d repertoire. J. Exp. Med. 171:1597.
30. Brenner, M. B., J. McLean, H. Scheft, R. A. Warnke, N. Jones, and
J. L. Strominger. 1987. Characterization and expression of the human ab T cell
receptor by using a framework monoclonal antibody. J. Immunol. 138:1502.
31. Hochstenbach, F., C. Parker, J. McLean, V. Gieselmann, H. Band, I. Bank,
L. Chess, H. Spits, J. L. Strominger, J. G. Seidman, and et al. 1988. Characterization of a third form of the human T cell receptor g/d. J. Exp. Med. 168:761.
GUINEA PIG CD1 GENE FAMILY