|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Immunology Unit, Department of Cellular
Biology and Pathology, University of Barcelona Medical School; Liver
Unit, Hematopathology Section, Laboratory of Anatomic Pathology, and
Department of Hematology, Hospital Clínic; and Institut
d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona,
Spain.
Ly-9 is a mouse cell-surface glycoprotein that is selectively
expressed on thymocytes and on mature T and B lymphocytes. Ly-9 belongs
to the CD2 subset of the immunoglobulin superfamily, an emerging family of cell signaling receptors. Recently, a
partial human Ly-9 complementary DNA (cDNA) sequence has been
described. Full-length cDNA clones were isolated that included the
initiation codon, the sequence encoding the full signal peptide, and 14 amino acids more in the cytoplasmic domain than in the previously
reported clone. The predicted extracellular domain of human Ly-9
contains 4 immunoglobulinlike domains, similar to those in mouse Ly-9. Northern blot analysis revealed that the human Ly-9 messenger RNA (2.6 kb) is expressed predominantly in lymph node, spleen, thymus, and
peripheral blood leukocytes. Four monoclonal antibodies (mAbs) were
raised against human Ly-9 by immunizing mice with the pre-B-cell line
300.19 stably transfected with human Ly-9 full-length cDNA. These mAbs
strongly stained the surfaces of cells transfected with human Ly-9 cDNA
but not of untransfected cells. Human Ly-9 expression was restricted to
T and B lymphocytes and thymocytes, with the highest levels of
expression on CD4+CD8 Several cell-surface molecules, termed accessory
molecules, cooperate with antigen-specific receptors by participating
in cell adhesion between lymphocytes and other cells or by contributing to cell signal transduction.1 A significant number of
these molecules belong to the immunoglobulin superfamily
(IgSF).2
Mouse Ly-9 is a cell-surface glycoprotein expressed on the cell
surfaces of thymocytes and mature B- and T-lymphocytes. It was
initially described as an alloantigenic marker of lymphocyte differentiation, and it has 2 alleles.3,4 Ly-9.1 is found in most mouse strains, whereas Ly-9.2 is only found in C57BL/6 and
related strains (C57BR, C57L, and C58).5,6 Because Ly-9 is
broadly expressed during lymphoid differentiation, monoclonal antibodies (mAbs) against the Ly-9.1 allotype are used as markers to
examine the contribution of embryonic stem-derived cells to lymphoid development in RAG-1- and RAG-2-deficient blastocyst complementation studies.7-9 These mAbs are also used to
test the percentage of chimerism in allophenic
mice.10
Ly-9 has also been reported to be a member of the IgSF with structural
features that place it within the CD2 family, which includes CD48,
CD58, 2B4, CD84, and CDw150.11,12 Members of this family
are expressed by various leukocytes and are involved in the activation
of lymphocytes and natural killer cells.13 Unlike other
CD2 family members, Ly-9 has 4 rather than 2 IgSF domains in its
extracellular part. Domains 1 and 3 are similar, as are domains 2 and
4, suggesting that Ly-9 arose from a progenitor with one V and one C2
domain.11 Ly-9 has the highest similarity to the
cell-surface molecules CD84 and CD48.14,15 The Ly-9 locus
lies close to the CD48, 2B4, CDw150,
and CD84 genes on both mouse and human chromosome 1. These
genes may be the result of gene duplication from a common ancestor,
with a second duplication event leading to the 4-domain structure of
Ly-9.14-16
The function of this leukocyte-specific cell-surface molecule is
unknown. However, analysis of the cytoplasmic tail of Ly-9 reveals
various motifs identical to those observed in several receptors
involved in cell signaling.17 The cytoplasmic tail of Ly-9
contains 2 unique tyrosine-based motifs (with the consensus amino acid
sequence TxYxxV/I) also found on the cytoplasmic domain of SLAM
(CDw150), 2B4, and CD84. This is critical for the binding of SLAM and
2B4 to a protein called SLAM-associated protein (SAP), which is mutated
in the immunodeficiency X-linked lymphoproliferative syndrome.18-20 It has been proposed that impaired
signaling by these receptors is responsible for this
immunodeficiency.13,18
Sandrin et al21 have reported a partial human Ly-9
complementary DNA (cDNA) sequence (HumLy-9) lacking the sequence
encoding the amino-terminal portion of the signal peptide and part of
the cytoplasmic tail. To date, human Ly-9 has not been serologically, biochemically, or functionally characterized. We have used several probes to isolate homologous full-length human Ly-9 clones. Mice were
immunized with cells transfected with the isolated cDNA to raise mouse
mAbs against human Ly-9. These mAbs were used to characterize the human
Ly-9 glycoprotein and its surface expression on normal and malignant cells.
Isolation of human and Ly-9
cDNA clones
RT-PCR of human Ly-9 from transfected cells and cell lines was
performed using the following oligos:
5'-GCTAGCATGAGTCAGCTGCAAATATTCTCT-3' and
5'-GCAGCAGCTGCTTTTCCTTTCAGGTGAA-3'. PCR products were cloned using the
TOPO subcloning kit (Invitrogen, Groningen, The Netherlands) and sequenced.
5' rapid amplification of cDNA ends procedure
Northern blot analysis Northern blot analysis was performed with nylon membranes from Clontech Laboratories (Multiple Tissue Northern Blot). Two micrograms poly A+ RNA from several tissues (spleen, lymph node, thymus, appendix, peripheral leukocytes, bone marrow, fetal liver, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas) was blotted after running on a denaturing formaldehyde 1.2% agarose gel. Northern blot membranes were hybridized with a 222-bp probe from immunoglobulinlike domain 1 of the human Ly9 clone and a 2000-bp human -actin cDNA as control probe. Hybridization was performed according to the manufacturer's instructions. Membranes were
autoradiographed for 72 hours (human Ly9) or 4 hours (-actin) at
 80°C using 2 intensifying screens.
Cells Cell lines were cultured in RPMI 1640 media (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS), L-glutamine, streptomycin, and penicillin. Cell lines Raji, Daudi, Jurkat, JM1, K562, HL60, RPMI, CESS, and U937 were from the American Tissue Culture Collection (Rockville, MD). The BEN cell line was obtained by the transformation of peripheral blood leukocytes with Epstein-Barr virus. Cultures of all cell lines were split the day before analysis. Blood leukocytes were isolated by Ficoll-Hypaque density gradient centrifugation of heparinized blood from healthy donors. Mononuclear cells were activated with 10 µg/mL phytohemagglutinin (Sigma, St Louis, MO), 5 ng/mL and 10 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma), and 500 nM calcium ionophore A231187 (Sigma) for 72 hours at a concentration of 2 × 106 cells /mL in RPMI 10% FCS. Erythrocytes were isolated from the red cell pellet after Ficoll-Hypaque sedimentation of blood. Thymus and tonsil lymphocytes were isolated as described.22,23 Bone marrow cells were obtained from 3 patients with anemia. Acute leukemias were classified on the basis of morphologic, cytologic, immunologic, and cytogenetic criteria described by French-American-British and European Group for the Immunological Classification of Leukemias (EGIL) groups and the World Health Organization (WHO).24 The diagnosis of lymphoproliferative disorders was based on the Revised European-American Classification of Lymphoid Neoplasms (REAL) and WHO classification.25All cells and tissues were obtained in accordance with protocols approved by the Ethics Committee of the Hospital Clínic of Barcelona. Informed consent was obtained from all the patients. Cell transfection Stably transfected cells were produced using the full-length human Ly-9 cDNA (1965 bp) cloned into the SmaI site of the pCI-neo expression vector (Promega) and the Ly-9 cDNA clone that lacked the third immunoglobulinlike domain. With the use of a Gene Pulser II Apparatus (Bio-Rad, Hercules, CA), 8 × 106 300.19 cells were transfected with 8µg linearized vector by electroporation (280 V, 950 µF). The cells were then plated in flat-bottomed 96-well tissue plates by limiting dilution, and stable transfectants were selected using 1.2 mg/mL G418 (Gibco-BRL). Sixteen clones from the transfection were examined by Northern blot, using a 222-bp fragment as probe. Clone 13, which expressed the highest level of message, was used in this study and was maintained in the presence of the selecting drug.COS cells were transfected with the full-length human Ly9 cDNA subcloned in the pCI-neo expression vector and human Ly9-Fc chimeric constructs according to the DEAE-dextran method.26 Cell-surface expression was examined after 48 hours by immunohistochemical analysis as described.27 Monoclonal antibodies HLy-9.1.25 (IgG1, ), HLy-9.1.38 (IgG1, ), HLy-9.1.77
(IgG2a, ), and HLy-9.1.84 (IgG1, ) mAbs were generated by fusing NS-1 myeloma cells with spleen cells from BALB/c mice immunized twice
with the mouse pre-B-cell line 300.19, stably transfected with the
full-length cDNA (clone 13). Hybridomas producing mAb reactive to
transfected but not untransfected cells were subcloned twice and used
to generate concentrated supernatant obtained from the culture of the
hybridomas in Integra CL 350 flasks (Integra Biosciences AG,
Wallisellen, Switzerland). Monoclonal antibody isotypes were determined
using the Isotyping Kit (Boehringer Mannheim). The mAbs were purified
using an Affi-Gel Protein A MAPS II kit (Bio-Rad, Hercules,
CA). Purified mAbs were biotinylated using Biotinamidocaproate
N-hydroxysuccinimide ester (Sigma). Other mAbs used in this study were
CD4-FITC, CD8-perCP, CD19-FITC, CD19-PE, CD19-perCP, CD38-PE,
CD56-FITC, CD16-PE, and CD3-APC (Becton Dickinson, San Jose, CA).
Fluorescein isothiocyanate (FITC)-conjugated antisera against human IgM
and mouse IgG were from Caltag (Burlingame, CA).
Immunofluorescence analysis Cells were kept at 4°C and examined immediately after isolation. Indirect immunofluorescence analysis of viable cells was carried out after the cells were washed twice (PBS containing 2% FCS and sodium azide 0.05%). Cells were then incubated for 20 minutes on ice, and each mAb was diluted to the optimal concentration for immunostaining. Isotype-matched murine mAbs that were unreactive with human leukocytes were used as negative controls. After they were washed, the cells were incubated for 20 minutes at 4°C with Avidin conjugated with FITC, phycoerythrin (PE), perCP, or Cy-Chrome (Pharmingen; Becton Dickinson). Single-, double-, triple-, and quadruple-color immunofluorescence analyses were performed on a FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells with forward and light scatter properties of monocytes and granulocytes were analyzed from the blood samples. At least 5000 cells per sample were analyzed.Cell-surface biotinylation and immunoprecipitation Cells (40 × 106) were washed 3 times in phosphate-buffered saline (PBS), resuspended in 800 µL saline, and surface-labeled with 200 µL biotin 1 mg/mL (Sigma) for 20 minutes at 4°C. After 1 wash in RPMI media with 10% FCS and 4 washes in PBS, the cells were lysed in 1 mL buffer containing 0.5% (vol/vol) NP40 and protease inhibitors as described.28 Immunoprecipitations were carried out using 15 µg mAbs. Cell lysates were precleared 3 times for 2 hours using 50 µL (50% vol/vol) mouse immunoglobulin-coated beads at 4°C. Cell lysates were precleared again overnight. The precleared lysate was then incubated with 50µL CD84 mAb-coated beads (50% vol/vol) or mouse immunoglobulin-coated beads with constant rotation at 4°C for 18 hours. Immunoprecipitates were washed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described.28 Samples were run in the presence or absence of 5% 2-mercaptoethanol. Mr was determined using prestained standard molecular weight markers (Gibco/BRL). Proteins were transferred by electroblotting with glycine transfer buffer (96 mM glycine, 12 mM Tris base in 10% methanol, pH 8.3) to PVDF membranes (Immobilon; Millipore, Boston, MA). After 2 hours of incubation at room temperature with blocking solution (10% nonfat milk in PBS), avidin peroxidase (100 ng/mL) (Sigma) was added for 1 hour. Membranes were washed with PBS with 0.1% Tween 20, and the blot was developed using enhanced chemiluminescence reagent (Amersham International, Little Chalfont, United Kingdom).
Isolation and sequencing of cDNA clones encoding human Ly-9 Four different cDNA clones were isolated from a gt11 cDNA
library constructed using RNA isolated from the human Raji cell line.
Only one of these clones, measuring approximately 2100 bp and
containing an entire sequence, was selected for further analysis. This
clone included the initiation codon and sequence encoding the full
signal peptide and 14 more amino acids in the cytoplasmic domain than
the previously reported clone.21 The sequence of the
selected clone revealed an ORF encoding a 608 amino acid protein with a
molecular mass of 65 kd after cleavage of the signal peptide. The amino
acid sequence was identical to mouse Ly-9 in 51% (73% if conserved
amino acid substitutions are considered) (Figure 1), showing that it is the human
homologue for this molecule. The leader peptide comprised 47 amino
acids, as in mouse Ly-9 cDNA (Figure 2).
Analysis of 50 clones obtained by 5' RACE from a human lymph node
library showed that all presented the same leader peptide (data
not shown).
The extracellular part of the molecule consisted of 4 immunoglobulinlike domains similar to those in mouse
Ly-9.12 The first and third were V-like domains that
lacked the usually conserved disulfide bonds between the Expression of human Ly-9 messenger RNA in several tissues Expression of human Ly-9 messenger RNA (mRNA) from several tissues was analyzed by Northern blot. An mRNA species of approximately 2.6 kb was detected in all lymphoid tissues tested, which included spleen, lymph node, thymus, appendix, peripheral leukocytes, and fetal liver, but not in bone marrow (Figure 3A-B). Slightly shorter bands, between 2.3 and 2.6 kb, might have represented an alternatively spliced mRNA. No hybridizing mRNA was detected in the heart, brain, lung, liver, skeletal muscle, or kidney (Figure 3). Placenta was the only nonlymphoid tissue with significant levels of mRNA.
Generation of mAb against human Ly-9 Four mAbs were obtained by immunizing mice with a clone of mouse pre-B-cell line 300.19 transfected with the full-length cDNA that was shown to contain the highest amount of mRNA by Northern blot. Each mAb reacted with 300.19 and COS transfected with human Ly-9; none reacted with untransfected parenteral cells (Figure 4, and data not shown).
Cell-surface staining of human cells, cell lines, and leukemias with anti-human Ly-9 monoclonal antibodies Anti-human Ly-9 mAbs were biotinylated and used to stain blood cells, lymphocytes isolated from thymus, bone marrow, blood and tonsil, and a large panel of lymphoid and myeloid cell lines. Anti-human Ly-9 mAb stained the cell surface of a third of the CD4+CD8+ double-positive thymocytes (Figure 5). The intensity of Ly-9 expression was higher on CD4+CD8 and
CD4CD8+ single-positive thymocytes. Some
CD4CD8 thymocytes expressed human Ly-9,
though according to 4-color flow cytometry, most of these cells
expressed CD3, whereas some staining was also seen on the
CD3CD4CD8 triple-negative
cells (Figure 6). Human Ly-9 cell-surface
expression correlated well with that of CD69. However, human Ly-9
appeared earlier than CD69 on the cell surfaces of the thymocytes
because a significant number of Ly-9-positive thymocytes did not
express CD69 (Figure 7).
Staining of peripheral blood leukocytes showed that human Ly-9
was expressed on mature lymphocytes but absent on monocytes, granulocytes, erythrocytes, and platelets, indicating that its expression was restricted to lymphocytes (Figure
8, and data not shown).
CD16+CD56+ natural killer cells expressed low
amounts of this protein (Figure 8). Mature T lymphocytes expressed
higher levels of human Ly-9 than B lymphocytes (Figure 8). Bone marrow
CD19 B cells expressed significant amounts of human Ly-9, but
expression was lower than that found on peripheral blood B lymphocytes
(Figure 8). Human Ly-9 expression significantly increased on the
surfaces of lymphocytes after 24-hour activation with PMA (data not
shown). Three- and 4-color immunofluorescence staining of tonsil
lymphocytes showed that the expression of human Ly-9 was higher on T
lymphocytes than on B lymphocytes (Figure
9). IgD
The Burkitt lymphoma B-cell lines (Daudi and Raji) and the Epstein-Barr
virus-transformed B-cell lines (CESS and BEN) expressed the highest
levels of Ly-9, whereas the rest of B-cell lines expressed low levels
(Table 1). Analysis of several samples of
B-cell malignancies, corresponding to various stages of B-cell
maturation, showed that human Ly-9 was expressed on most of them (Table
2), indicating that human Ly-9 is a
pan-B-cell marker. T-cell line Jurkat also expressed significant
amounts of human Ly-9. Myelomonocytic cell lines HL60 and U937 and
erythromyeloid cell line K562 were negative for Ly-9 (Table 1).
However, half the acute myeloblastic leukemias tested were positive for
Ly-9 (Table 2).
Immunoprecipitation of human Ly-9 from 300.19 cells transfectants and B cell-line Daudi with anti-human Ly-9 monoclonal antibodies We performed immunoprecipitations from biotinylated untransfected or human Ly-9 cDNA-transfected 300.19 cells using the anti-human Ly-9 mAb (Figure 10). The mAb precipitated a single band of approximately 120 kd in reducing and nonreducing conditions. In contrast, when the same antibodies were used to precipitate human Ly-9 from the B-cell line Daudi, 2 bands of 120 kd and 100 kd were seen (Figure 10A). The second band was similar to that detected when 300.19 cells were transfected with the isoform that lacks the fourth immunoglobulinlike domain. To test whether the second band found in Daudi cells corresponded to an alternatively spliced form of Ly-9, RT-PCR was performed with RNA isolated from Daudi cells. Two major bands were detected (Figure 10B), and, after subcloning and sequencing, they were shown to encode the full-length cDNA and an alternatively spliced isoform lacking the fourth immunoglobulinlike domain, respectively. The sequence of 20 independent clones of the lower band showed that they all lacked the sequence corresponding to the fourth domain. Thus, the spliced variant isolated from the cDNA might have been expressed on the surface of certain B cells.
This paper describes the cloning and characterization of the human surface molecule Ly-9. We report the isolation of cDNA encoding full-length human Ly-9 from a human Raji cDNA library. Unlike the recently reported human Ly-9 cDNA sequence,21 our cDNA sequence contains the complete leader sequence and part of the cytoplasmic domain. In contrast with the published sequence, our results show that mouse and human leader peptides have the same number of amino acids (Figures 1, 2). Although the predicted leader peptide of both human and mouse Ly-9 is long, some other cell-surface protein, including CD49e, CD62P, CD163, and CD129, have been shown to contain long leader peptides of 40 or more amino acids. The number of amino acids of the cytoplasmic tail is identical in mouse
and human, except for one amino acid.11,29 The lack of 14 amino acids in the cDNA reported elsewhere may be due to mRNA
splicing.29 Whether the presence of a spliced variant of
the cytoplasmic tail has any biologic significance remains unclear.
However, it may have, because the spliced sequence contains 2 threonines, 2 serines, and 1 tyrosine in a YxxL putative SH2-binding motif (Figure 2). Two other tyrosine residues in the cytoplasmic tail
of human and mouse Ly-9 are in a motif in agreement with the consensus
sequence T- Northern blot analysis showed the expression of a 2.6-kb Ly-9 mRNA in lymphoid tissues. The pattern of expression is consistent with that of mouse Ly-9, which is found on mature T and B lymphocytes and thymocytes.3-5 Slightly differently sized bands between 2.3 and 2.6 kb were observed in most lymphoid tissues and might represent alternatively spliced mRNA encoding several isoforms of the molecule. Four mAbs were generated by immunizing mice with a stable transfectant of human Ly-9. These mAbs stained 300.19 pre-B-cell line transfected with human Ly-9 cDNA but did not stain untransfected 300.19 cells. Using these mAbs, we showed that human Ly-9 protein was expressed on the cell surfaces of thymocytes. The expression of Ly-9 seems to have preceded positive selection during T-cell differentiation because Ly-9 appeared before CD69. CD69 is known to appear on thymocyte surfaces that have undergone positive selection.30 In addition, peripheral blood and tonsil T and B lymphocytes expressed significant levels of human Ly-9. The intensity of expression of human Ly-9 on B cells was lower than on T lymphocytes. Bone marrow B cells expressed low levels of human Ly-9, indicating that its expression increased with cell maturation. Most B-cell lines, leukemias, and lymphomas expressed human Ly-9. All multiple myeloma samples analyzed were positive for Ly-9, indicating that its expression is not lost during differentiation to plasma cells, whereas most differentiation antigens expressed on B cells were no longer expressed on terminally differentiated plasma cells. Monocytes, granulocytes, erythrocytes, and platelets did not express significant amounts of human Ly-9. Overall, these results suggest that the expression of human Ly-9 is restricted to lymphocytes. However, a significant number of acute myeloblastic leukemias expressed human Ly-9, indicating that some cells of the myeloid lineage also express human Ly-9. The anti-human Ly-9 mAbs specifically immunoprecipitated a
single polypeptide of 120 kd from the cells transfected with
full-length cDNA. However, when Ly-9 was immunoprecipitated from the
B-cell line Daudi, 2 bands of 120 kd and 100 kd could be observed. The smaller band is probably a spliced form of the molecule lacking the
fourth immunoglobulinlike domain. A band of similar size was observed
when anti-human Ly-9 mAbs were used to immunoprecipitate this protein
with cells transfected with the cDNA of clone HLy-9 Thymocytes and B and T lymphocytes express a previously undescribed glycoprotein representing the human homologue of mouse Ly-9. This protein can be used as a surface marker to study lymphocyte differentiation. The mAbs generated in this study may improve our understanding of the pattern of expression of Ly-9 and the delineation of the cytoplasmic and cell-surface molecules with which it may interact. These mAbs will be useful to establish hypotheses regarding the biologic roles of this molecule in lymphocyte development and activation.
We thank Isabel Sánchez for assistance in these experiments and Dr M. Streuli for providing 300.19 cells.
Submitted August 31, 2000; accepted January 31, 2001.
Supported by grants SAF97-0136 and SAF00-0037 from the Comisión Interministerial de Ciencia y Tecnología. V.T. is a Fellow of the Programa Nacional de Formación de Personal Investigador.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Pablo Engel, Immunology Unit, Department of Cellular Biology and Pathology, Facultad de Medicina, C/Casanovas 143, Barcelona E 08036, Spain; e-mail: engel{at}medicina.ub.es.
1. Weiss A, Littman DR. Signal transduction by lymphocyte antigen receptors. Cell. 1994;76:263-274[CrossRef][Medline] [Order article via Infotrieve]. 2. Williams A-F, Barclay AN. The immunoglobulin superfamily-domains for cell surface recognition. Ann Rev Immunol. 1998;88:381-405. 3. Ledbetter JA, Golding JW, Tsu TT, Herzenberg LA. A new mouse lymphoid alloantigen (Lgp100) recognized by a monoclonal rat antibody. Immunogenetics. 1979;8:347-360[CrossRef]. 4. Mathieson BJ, Sharrow SO, Bottomly K, Fowlkes BJ. Ly-9, an alloantigenic marker of lymphocyte differentiation. J Immunol. 1980;125:2127-2136[Abstract]. 5. Hogarth PM, Graig J, McKenzie I-FC. A monoclonal antibody detecting the Ly-9.2 (Lgp-100) cell-membrane alloantigen. Immunogenetics. 1980;11:65-74[CrossRef][Medline] [Order article via Infotrieve]. 6. Kozak CA, Davidson WF, Morse HC III. Genetic and functional relationship of the retroviral and lymphocyte alloantigen loci on mouse chromosome 1. Immunogenetics. 1984;19:163-168[CrossRef][Medline] [Order article via Infotrieve].
7.
Arroyo AG, Yang JT, Rayburn H, Hynes RO.
Differential requirements for 8. Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt F-W, Orkin SH. The T cell leukemia oncoprotein SCL/tal-1 essential for development of all hematopoietic lineages. Cell. 1996;86:47-57[CrossRef][Medline] [Order article via Infotrieve].
9.
Potocnik AJ, Kohler H, Eichmann K.
Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells.
Proc Natl Acad Sci U S A.
1997;94:10295-10300
10.
Shechler JMG, Lawler A, Hartley JW, et al.
Induction of murine acquired immunodeficiency syndrome (MAIDS) in allophenic mice generated from strains susceptible and resistant to disease.
J Exp Med.
1996;184:2101-2108 11. Sandrin MS, GumLey TP, Henning MM, et al. Isolation and characterization of cDNA clones for mouse Ly-9. J Immunol. 1992;149:1636-1641[Abstract]. 12. Davis SJ, van der Merwe PA. The structure and ligand interactions of CD2: implications for T-cell function. Immunol Today. 1996;617:177-187. 13. Tangye SG, Phillips JH, Lanier LL. The CD2-subset of the Ig superfamily of cell surface molecules: receptor-ligand pairs expressed by NK cells and other immune cells. Semin Immunol. 2000;12:149-157[CrossRef][Medline] [Order article via Infotrieve].
14.
De la Fuente MA, Pizcueta P, Nadal M, Bosch J, Engel P.
CD84 leukocyte antigen is a new member of the Ig superfamily.
Blood.
1997;90:2398-2405 15. De la Fuente MA, Tovar V, Pizcueta P, Nadal M, Bosch J, Engel P. Molecular cloning, characterization and chromosomal localization of the mouse homologue of CD84 a member of the CD2 family of cell surface molecules. Immunogenetics. 1999;49:249-255[CrossRef][Medline] [Order article via Infotrieve]. 16. Kingsmore SF, Souryal CA, Watson M-L, Patel DD, Seldin M-F. Physical and genetic linkage of the genes encoding Ly-9 and CD48 on mouse and human chromosome 1. Immunogenetics. 1995;42:59-62[CrossRef][Medline] [Order article via Infotrieve].
17.
Songyang Z, Shoelson SE, McGlade J, et al.
Specific motifs recognized by SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, Syk, and Vav.
Mol Cell Biol.
1994;14:2777-2785 18. Sayos J, Wu C, Morra M, et al. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature. 1998;395:462-469[CrossRef][Medline] [Order article via Infotrieve].
19.
Tangye SG, Lazetic S, Woollatt E, Sutherland GR, Lanier LL, Phillips JH.
Human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHIP-2 and the adaptor signaling protein SAP.
J Immunol.
1999;162:6981-6985
20.
Nichols KE, Harkin DO, Levitz S, et al.
Inactivating mutations in a SH-2 domain-encoding gene in X-linked lymphoproliferative syndrome.
Proc Natl Acad Sci U S A.
1998;95:13765-13770 21. Sandrin MS, Henning MM, Lo MF, Baker E, Sutherland GR, McKenzie IFC. Isolation and characterization of cDNA clones for HumLy9: the human homologue of mouse Ly9. Immunogenetics. 1996;43:13-19[Medline] [Order article via Infotrieve]. 22. Vanhecke D, Leclercq G, Plum J, Vanderckhove B. Characterization of distinct stages during the differentiation of human CD69+CD3+ thymocytes and identification of thymic emigrants. J Immunol. 1995;155:1862-1872[Abstract]. 23. Ingles J, Engel P, de la Calle O, Gallart T. Differential responsiveness of human B lymphocytes to phorbol ester and calcium ionophore based on their state of activation. Immunology. 1989;67:359-364[Medline] [Order article via Infotrieve]. 24. Rothe G, Schitz G. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies: working group on flow cytometry and image analysis. Leukemia. 1996;10:877-895[Medline] [Order article via Infotrieve]. 25. Harris NL, Jaffe ES, Stein H, et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994;1361-1392. 26. Engel P, Nojima I, Rothstein D, et al. The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils and monocytes. J Immunol. 1993;150:4719-4732[Abstract].
27.
Engel P, Wagner N, Miller A, Tedder TF.
Identification of the ligand binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid-dependent ligand.
J Exp Med.
1995;181:1581-1586
28.
Tedder TF, Schlossman SF.
Phosphorylation of the B1 (CD20) cell surface molecule expressed by normal and malignant human B lymphocytes.
J Biol Chem.
1988;263:10009-10015 29. Tovar V, de la Fuente MA, Pizcueta P, Bosch J, Engel P. Gene structure of the mouse leukocyte cell-surface molecule Ly9. Immunogenetics. 2000;51:788-793[CrossRef][Medline] [Order article via Infotrieve].
30.
Yamashita I, Nagata T, Tada T, Nakayama T.
CD69 cell surface expression identifies developing thymocytes which augmentation for T cell antigen receptor-mediated positive selection.
Int Immunol.
1993;5:1139-1150
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  X. Romero, N. Zapater, M. Calvo, S. G. Kalko, M. A. de la Fuente, V. Tovar, C. Ockeloen, P. Pizcueta, and P. Engel CD229 (Ly9) Lymphocyte Cell Surface Receptor Interacts Homophilically through Its N-Terminal Domain and Relocalizes to the Immunological Synapse J. Immunol., June 1, 2005; 174(11): 7033 - 7042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Martin, J. M. Del Valle, I. Saborit, and P. Engel Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229 J. Immunol., May 15, 2005; 174(10): 5977 - 5986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Graupera, S. March, P. Engel, J. Rodes, J. Bosch, and J.-C. Garcia-Pagan Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers Am J Physiol Gastrointest Liver Physiol, April 1, 2005; 288(4): G763 - G770. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Chtanova, S. G. Tangye, R. Newton, N. Frank, M. R. Hodge, M. S. Rolph, and C. R. Mackay T Follicular Helper Cells Express a Distinctive Transcriptional Profile, Reflecting Their Role as Non-Th1/Th2 Effector Cells That Provide Help for B Cells J. Immunol., July 1, 2004; 173(1): 68 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Del Valle, P. Engel, and M. Martin The Cell Surface Expression of SAP-binding Receptor CD229 Is Regulated via Its Interaction with Clathrin-associated Adaptor Complex 2 (AP-2) J. Biol. Chem., May 2, 2003; 278(19): 17430 - 17437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Howie, S. Okamoto, S. Rietdijk, K. Clarke, N. Wang, C. Gullo, J. P. Bruggeman, S. Manning, A. J. Coyle, E. Greenfield, et al. The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production Blood, September 26, 2002; 100(8): 2899 - 2907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Morra, M. Simarro-Grande, M. Martin, A. S.-I. Chen, A. Lanyi, O. Silander, S. Calpe, J. Davis, T. Pawson, M. J. Eck, et al. Characterization of SH2D1A Missense Mutations Identified in X-linked Lymphoproliferative Disease Patients J. Biol. Chem., September 21, 2001; 276(39): 36809 - 36816. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
sheets.
The second and fourth had the structural features of an IgSF-truncated
C2 set domain with 2 putative disulfide bonds. One of the isolated
clones (HLy-9 no. 2) lacked the sequence corresponding to the fourth
immunoglobulinlike domain (Figure 
4), and 300.19 untransfected cells were immunoprecipitated with the HLy-9.1.25 mAb and
analyzed under reducing and nonreducing conditions on a 10% sodium
dodecyl sulfate-polyacrylamide gel. Molecular weights (kd) were
determined by the migration of a known protein standard. (B) cDNA was
made with mRNA from 300.19 cells transfected with full-length human
Ly-9 cDNA, Ly-9 cDNA lacking the fourth immunoglobulinlike domain
(HLy-9-
-Y-X-X-V/I for the binding of SAP and SHP-2, found in
SLAM and 2B4.