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J Biol Chem, Vol. 275, Issue 5, 3431-3437, February 4, 2000
From Immunex Corp., Seattle, Washington 98101
CD7 is a 40-kDa protein found primarily on T, NK,
and pre-B cells; the function of the CD7 protein in the immune system
is largely unknown. The K12 (SECTM1) protein was originally identified by its location just upstream of the CD7 locus. The
K12 gene encodes a transmembrane protein of unknown
function. In order to clone a K12-binding protein, we generated a
soluble version of the human K12 protein by fusing its extracellular
domain to the Fc portion of human IgG1. Flow cytometry
experiments showed that the K12-Fc fusion protein bound at high levels
to both human T and NK cells. Precipitation experiments using K12-Fc on
35S-radiolabeled NK cells lysates indicated that the K12
cognate was an approximately 40-kDa protein. A human peripheral blood T
cell cDNA expression library was screened with the K12-Fc protein, and two independent, positive cDNA clones were identified and sequenced. Both cDNAs encoded the same protein, which was CD7. Thus, K12 and CD7 are cognate proteins that are located next to each
other on human chromosome 17q25. Additionally, we have cloned the gene
encoding the mouse homologue of K12, shown that it maps near the mouse
CD7 gene on chromosome 11, and established that the mouse
K12 protein binds to mouse, but not human, CD7. Mouse K12-Fc inhibited
in a dose-dependent manner concanavalin A-induced proliferation, but not anti-TcR The K12 (SECTM1) gene was originally
identified (1) as being directly 5' of the locus encoding the human
CD7 gene on human chromosome 17 (2). The 3' end of the
K12 gene is about 5 kilobases upstream of the start of the
human CD7 gene; both genes are transcribed in the same
direction (1). The human K12 protein has been shown to be primarily
expressed in spleen, prostate, testis, small intestine, and in
peripheral blood leukocytes (1). Several features of the protein
encoded by the K12 gene suggested to its discoverers that it
might be cytokine-like. One feature is that K12 encodes a transmembrane
protein, a trait that is shared with a number of growth factors
including flt3 ligand (3, 4), c-kit ligand (5-7), and
colony stimulating factor 1 (8). The other notable feature of K12 is
that the extracellular domain is similar in some, but not all, respects
to an immunoglobulin-like domain. However, immunoglobulin-like domains
in proteins are generally associated with receptors for cytokines
(e.g. c-kit, KDR, FGFR), not the cytokines themselves.
We have cloned the cognate of K12 to establish what its biological
function might be, and discovered that K12 is a binding partner for
CD7, the protein encoded by its neighboring gene. We have also cloned
the mouse homologue of the K12 gene, found that it binds to
mouse CD7, but not human CD7, and mapped its location near the mouse
CD7 gene on mouse chromosome 11. These studies lay the
groundwork for determining the activities as well as the interactions
of CD7 and K12 in the immune system.
Cloning of the Human and Mouse K12 and CD7 Genes--
The human
K12 protein was cloned based on the published sequence (1) using
reverse transcriptase-PCR1
from mRNA prepared from the K562 erythroleukemia cell line. A mouse
protein related to the human K12 sequence was identified as
an EST (AA734402) from a proximal colon cDNA library. The EST was
purchased and sequenced in its entirety. Encoded within the cDNA is
a 212-amino acid transmembrane protein that shares 36% overall amino
acid identity over its entire length with the human K12 protein.
The mouse CD7 gene was cloned using PCR from an EL4.6 Generation of K12-Fc Fusion Proteins--
Both the human and
mouse K12-Fc fusion proteins were made by using Sew-PCR to attach the
Fc portion of human IgG1 to that part of the gene encoding the
extracellular domain of K12 (amino acids 1-145 in the human clone (1),
amino acids 1-160 in the mouse clone). The fusion proteins were
transiently expressed in CV-1/EBNA cells and purified from the
conditioned medium using protein-A-Sepharose (Amersham Pharmacia Biotech).
Cloning of the K12 Cognate from a Human PBT cDNA Expression
Library--
The human peripheral blood T cell library cDNA
expression library was constructed in the pDC409 vector using methods
previously described (11) and contains about 0.5 × 106 cDNA clones. Approximately 78% of clones in the
library contain inserts, and the average insert size is about 1.2 kilobases. The human K12-Fc fusion protein was used to screen the
library essentially as described previously (3). Two positive pools of
approximately 1600 cDNAs each were identified. These positive pools
were subdivided into smaller and smaller groups until individual
cDNAs could be picked and tested. Once individual clones were
identified, full double stranded sequencing of the clones was obtained.
Precipitation of a K12-Fc-binding Protein from NK
Cells--
Primary human NK cells (1 × 106 cells/ml)
were radiolabeled overnight with 50 µCi/ml
[35S]cysteine/methionine) (ProMix, Amersham Pharmacia
Biotech). Radiolabeled cells were lysed with 1 ml of RIPA E lysis
buffer (PBS, 1% Triton). 150 µl of lysate were incubated with 1 µg
of human K12-Fc or a control Fc fusion protein for 1 h at 4 °C.
Precipitated proteins were collected onto Protein-A-Sepharose and
loaded separated on a 4-20% Tris glycine gel (Novex, San Diego, CA)
under denaturing, reducing conditions. The gel was fixed, treated with
Amplify (Amersham Pharmacia Biotech), dried, and exposed to XAR-5 film.
K12/CD7 Binding Studies--
COS-1 cells were transfected with
full-length human CD7, mouse CD7, or vector only
cDNA using DEAE dextran. Two days post-transfection the cells were
assayed for the capacity to bind human K12-Fc or mouse K12-Fc as
described previously (3) with the following modifications. The binding
media (RPMI 1640, 1% FBS, 0.02% sodium azide, 20 mM
HEPES, pH 7.2) was modified to include 1 mM
MnCl2. In some experiments, the transfected cells were
incubated with zero, 20 µg/ml, or 2 mg/ml of ConA in BM/MM prior to
binding with the Fc proteins. Following incubation for 30 min at room
temperature and the continued presence of ConA, 1 µg/ml of either
human K12-Fc or mouse K12-Fc was then added to the appropriate slides.
Binding of the Fc protein was detected with 125I-mouse
anti-human Fc antibody (Amersham Pharmacia Biotech) as described
previously (3). After binding the iodinated antibody, the cells were
washed and then the radioactivity quantified by Storm PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Chromosomal Location of the Mouse K12 Gene--
The chromosomal
location of the putative mouse K12 protein was determined using the
Jackson Laboratory radiation hybrid panel mapping resource (12).
Human NK Cell Preparation--
Human NK cells were isolated from
the peripheral blood of normal human donors. Two hundred ml of
heparinized blood were collected by venipuncture, then diluted 1:1 with
room temperature PBS. Forty ml of blood solution was underlayered with
10 ml of Isolymph (Gallard-Schlesinger, Carle Place, NY), and
peripheral blood mononuclear cells were isolated by density gradient
centrifugation for 20 min at 2200 rpm. The interface containing the
peripheral blood mononuclear cells was removed and diluted 1:1 in room
temperature PBS. The samples were spun for 15 min at 1800 rpm. The
pellets were then washed twice with PBS, spun 10 min at 1500 rpm, and
resuspended in RPMI containing 5% FBS.
Adherent cells were removed from the peripheral blood mononuclear cells
by incubation at less than 5 × 106/ml for 1 h at
37 °C in a T175 tissue culture flask in RPMI containing 5% FBS
(supplemented with 1 mM sodium pyruvate, 550 nM
L-arginine, 272 nM L-asparagine,
13.6 nM folic acid, 10 mM Hepes, 20 µg/ml gentamycin, 50 nM 2-mercaptoethanol, and 0.210 mg/ml
penicillin/streptamycin/glutamine).
Further enrichment for NK cells then occurred by incubating the
non-adherent peripheral blood mononuclear cell fractions with anti-CD3-biotin, anti-CD19-biotin, and anti-HLA-DR-biotin for 1 h
at room temperature. The antibody-coated cells were then washed and
incubated with 15 µl of streptavidin/Dynabeads (Dynal, Oslo, Norway)
per 10 × 106 cells, with constant gentle agitation
for 20 min at room temperature. The beads were then removed (with their
attached cells) using a Dynal magnet. This negative selection process
was repeated once more, resulting in a highly enriched NK cell
population, as identified by FACS (75-92% CD56+,
CD16+).
NK Activation Assays and Flow Cytometry--
Enriched NK cells
were cultured in 6-well plates that had previously been coated with
human and/or murine IgG, human CD7 mAb (clone 8118.1) or human K12-Fc
at 2.5 µg/ml in PBS. The human NK cells were added at 2 × 106/ml in RMPI containing 5% FBS and incubated for 20 h at 37 °C in 5% CO2.
Cells were harvested by aspiration and washed in PBS containing 2% FBS
and 0.1% azide, then resuspended in a FACS blocking buffer of PBS
containing 10% FBS, 10% normal goat serum, 10% normal rabbit serum,
and 0.1% azide. A maximum of 1 × 106 cells was
incubated with the indicated mAb-fluorochrome conjugates for 1 h
at 4 °C in a total volume of 100 µl. The cells were then washed in
2 ml of PBS containing 2% FBS and 0.1% azide and resuspended in 300 µl of PBS containing 2% FBS, 0.1% azide, and propidium iodide.
Following resuspension, the cells were analyzed for fluorescent antibody binding on a FACScan flow cytometer using Cellquest Software (Becton Dickinson, Franklin Lakes, NJ).
Antibodies--
Antibodies directed against human CD7 were
purchased from the following sources: clone M-T701, Pharmingen, San
Diego, CA; clone 8118.1, Immunotech, Westbrook, ME; clone 4H9, Becton
Dickinson; and clones RFT-2a, WM31, and CLB-3A1, Research Diagnostics,
Inc., Flanders, NJ. Other antibodies used were: PE-conjugated
anti-huCD69 (clone FN50), PE-conjugated anti-huCD25 (clone M-A251),
PE-conjugated anti-huCD56 (clone B159), PE-conjugated IgG1 control,
biotin-labeled anti-huCD3 (clone UCHT1), and biotin-labeled anti-huCD19
(clone B43), all from Pharmingen (San Diego, CA), and PE-conjugated
anti-huCD54 (clone 84H10), fluorescein isothiocyanate-conjugated
anti-huCD16 (clone 3G8), biotin-labeled anti-huHLA-DR (clone B8.12.2)
all from Immunotech (Westbrook, ME). Purified human IgG was obtained from Sigma, and purified mouse IgG was obtained from Caltag
(Burlingame, CA).
Identification of CD7 as the Human K12 Cognate--
To identify
with which molecule(s) the K12 protein might interact, we made a
protein comprising the extracellular domain of human K12 (amino acids
1-145) fused in-frame with the Fc portion of human IgG1. The K12-Fc
fusion protein was then used in flow cytometry experiments to determine
which cell types might express on their cell surface a binding partner,
or cognate, for the K12 protein. High levels of K12-Fc binding were
detected on primary human T cells, either resting cells or cells
treated with ConA (Fig. 1, panels
A and B). Human NK cells also displayed high levels of
K12-Fc binding (data not shown). In contrast, a number of other human
and mouse cell lines failed to bind the K12-Fc protein (data not
shown).
Primary human NK cells were radiolabeled with
[35S]cysteine/methionine for 4 h, then washed,
lysed, and incubated with the K12-Fc fusion protein in an effort to
precipitate a cognate protein. A single protein band of approximately
40 kDa was precipitated from the NK cells (Fig.
2), suggesting that expression cloning might be a viable way to identify the K12 cognate.
As a result of the T cell binding data, we screened a cDNA
expression library made from human peripheral blood T cells that had
been stimulated with PHA. Fifty pools of approximately 1600 cDNAs
each were transfected into CV-1/EBNA cells, and 2 days later the
transfected cells were tested for their capacity to bind the K12-Fc
fusion protein. Two positive cDNA pools (numbers 85 and 129) were
found that conferred on the CV-1/EBNA cells the capacity to bind
K12-Fc. These cDNA pools were subsequently subdivided into smaller
and smaller groups until single positive cDNA clones were isolated
from each original pool. Sequencing of these cDNAs and comparison
with public DNA data bases revealed that the cDNA from each
positive pool encoded a full-length clone of the human CD7
gene. The size of the protein precipitated from NK cell lysates (40 kDa) is consistent with the reported size of human CD7 (13, 14).
Monoclonal Antibodies to Human CD7 Block Binding of
K12-Fc--
Six commercial antibodies to the extracellular domain of
human CD7 were purchased and tested for their capacity to block the binding of K12-Fc to Jurkat cells, which express high levels of CD7.
The antibodies blocked the binding of the K12-Fc fusion protein to the
cells to varying degrees (Table I). Along
these same lines, the human K12-Fc fusion protein blocked the binding
of each member of the panel of monoclonal antibodies to CD7 (Table
I).
Cloning of the Mouse Homologue of the Human K12 Gene--
The
human K12 amino acid sequence was compared with amino acid translations
of both public and proprietary EST data bases in an effort to identify
a mouse homologue of K12. A single EST sequence was identified
(AA734402) from a mouse proximal colon library that encoded a protein
that appeared to be related to human K12. The EST was
purchased and sequenced in its entirety. Encoded within the cDNA is
a 212-amino acid transmembrane protein that overall shares 36% amino
acid identity over its entire length with the human K12 protein (Fig.
3). Focusing on just the extracellular regions, the amino acid identity between these putative human and mouse
homologues is 44%. A second EST clone (971012tram001354ht) from a
proprietary high throughput sequencing project contained essentially
the same sequence as AA734402. One key difference was that this second
cDNA contained an apparent intron after amino acid residue 134. This position corresponds to the position of an intron within amino
acid 135 of the human K12 protein (15), and supports the idea that the
putative mouse K12 protein is either the true mouse homologue of the
human protein, or is a closely related family member.
In contrast to the human K12 protein, which has a single potential site
for N-linked glycosylation, the putative mouse K12 protein
contains four such sites, one of which is conserved in the location of
the single glycosylation site in the human protein (Fig. 3). The
position of two cysteine residues in the extracellular domains of human
and mouse K12 also appear to be conserved. The sequence of the human
K12 cytoplasmic domain contains a di-acidic signal (DXE) required for
selective export from the endoplasmic reticulum (16). However, the
cytoplasmic domain of the putative mouse K12 protein does not contain
this motif.
A mouse K12-Fc fusion protein was constructed from the extracellular
domain of the mouse gene and was tested for its capacity to bind to
mouse lymph node T cells (Fig. 4,
panels A and B). The mouse K12-Fc fusion protein
specifically bound to the mouse lymph node T cells, although the
intensity of the binding was not nearly as strong as seen with human
K12-Fc on human peripheral blood T cells (Fig. 1, panels A
and B).
Chromosomal Location of the Mouse K12 Gene--
The human
K12 gene is adjacent to the human CD7 gene on
human chromosome 17 (1). The chromosomal location of the putative mouse
K12 gene was determined by radiation hybrid mapping (12). The mouse K12 gene is localized just proximal to the
CD7 locus on mouse chromosome 11 (17) with a LOD of 21.0 (The Jackson Laboratory, Bar Harbor, ME). Thus, in mice, as in humans,
the K12 gene is located near the CD7 gene.
Cross-species Binding of Human or Mouse K12-Fc to Human and Mouse
CD7--
As noted above, human K12 and the putative mouse K12 proteins
share only 44% amino acid identities in their extracellular domains.
Similarly, human and mouse CD7 are 54% identical overall, but share
only 49% identity in their extracellular domains (analyzed by the GCG
GAP program). Thus, the percentage of amino acid sequence identity of
the extracellular domains of mouse and human K12 proteins (44%) is
very similar to that seen between the extracellular domains of mouse
and human CD7 proteins (49%). To determine whether mouse K12-Fc binds
to mouse CD7, PCR was used to clone the full-length mouse
CD7 cDNA into an expression vector, which was then
transfected into COS-1 cells. The transfected cells were then tested
for their capacity to bind either human or mouse K12-Fc fusion
proteins. The human K12-Fc protein binds strongly to cells transfected
with the human CD7 cDNA, but does not bind to the
surface of cells transfected with the mouse CD7 cDNA
(Fig. 5). In contrast, mouse K12-Fc bound
to transfected cells expressing the mouse CD7 protein, but was not
capable of binding to human CD7 (Fig. 5). Thus, K12 and CD7 proteins
bind each other in a species-specific manner.
The human K12-Fc fusion protein was radiolabeled and used in binding
experiments to determine its affinity for Jurkat cells (a human T cell
leukemia cell line) or KG-1 cells (a human myelogenous leukemia cell
line), both of which express CD7. In preliminary experiments, the
binding affinity (Ka) of human K12-Fc for human CD7
was estimated to be in the range of 1 × 108
M Expression of the K12 Protein on the Cell
Surface--
Slentz-Kesler and co-workers (1) reported that the human
K12 protein was found both inside the cell and in medium conditioned by
the cells, but that it was not seen on the cell surface. We examined
the capacity of soluble mouse and human CD7-Fc fusion proteins (made by
fusing the full-length extracellular domains of the proteins to the Fc
region of human IgG1) to bind to cells transfected with
either mouse or human full-length K12 cDNAs (Fig. 6). Human CD7-Fc bound to the surface of
cells transfected with a full-length human K12 cDNA, and
mouse CD7-Fc bound to cells transfected with a full-length mouse
K12 cDNA. No cross-species binding was observed. These
experiments show that the K12 protein can be expressed on the cell
surface, at least on transfected cells. However, using flow cytometry
with human CD7-Fc we confirmed the finding (1) that K12 protein is not
detectable on the surface of the K562 and MDA-231 cell lines, even
though these cells express K12 mRNA (data not
shown).
K12-Fc Blocks ConA, but Not Anti-TcR, Induced Cell
Proliferation--
The mouse K12-Fc fusion protein was tested for its
capacity to inhibit the proliferation of BALB/c lymph node cells that
had been stimulated with either ConA (Fig.
7A) or immobilized anti-TcR K12-mediated CD7 Cross-linking on Human NK Cells Enhances Surface
Molecule Expression--
The capacity of K12 to induce human NK cell
activation through its interaction with CD7 was analyzed by examining
the expression of surface molecules associated with cellular
activation. Flow cytometric analysis showed that resting NK cells
cultured with immobilized human and mouse IgG expressed very low levels
of CD25, CD69, and the adhesion molecule, CD54. After overnight culture with immobilized anti-CD7, increases in both CD25 and CD54 expression on NK cells were observed (Table II).
Overnight culture of NK cells with immobilized human K12-Fc resulted in
increases in CD25, CD69, and CD54 expression (Table II). The increase
in surface molecule expression induced by K12-Fc-mediated cross-linking
of CD7 ranged between 4-6-fold over control, and the increase induced by anti-CD7 mAbs ranged between 2-4-fold over control. The activity of
the K12-Fc and the anti-CD7 mAbs on NK cells are roughly equivalent on
a molar basis at the concentrations tested. CD7 cross-linking also
increased CD69 expression in some experiments (data not shown). Addition of anti-CD7 antibodies or K12-Fc in solution (not immobilized) to NK cultures did not result in enhancement of cell surface molecule expression (data not shown).
The major finding of this paper is the demonstration that the K12
protein is the counterstructure for CD7. What is the biological significance, if any, of K12 binding to CD7? This is the key question to which we do not yet have an answer. It is not clear if the interaction between these proteins is meant to stimulate cells expressing K12, cells expressing CD7, or both. Signaling through CD7
has been reported using anti-CD7 antibodies (18-20), indicating that
the cytoplasmic domain of CD7 must contain some signal transducing elements, or that it complexes with a protein(s) containing such elements. If the K12 protein is not expressed on the cell surface, as
has been suggested (1), the simplest interpretation of the data is that
a soluble form of the K12 protein stimulates CD7 on T, NK, or some
other cell type. We have shown, however, that the K12 protein can be
expressed on the cell surface, at least in transfected cells (Fig. 6).
Therefore, we remain open to the possibility that the CD7 protein could
trigger some signaling pathway in cells expressing K12 on the cell
surface. Unlike other situations where a growth factor and a growth
factor receptor can be readily defined on the basis of size and
function (e.g. EGF and the EGF receptor), the situation here
is less clear. The binding partners are nearly identical in size (248 amino acids for human K12; 240 amino acids for human CD7), so neither
fits the classical definition of a cell surface receptor, which is usually significantly larger than its ligand.
The finding that the CD7-K12 genes are directly adjacent to
each other (1) is the first instance of which we are aware of a pair of
cognate proteins being so closely linked at the genomic level. Although
the mouse and human K12 genes are only 36% identical at the
amino acid level, we believe that the mouse protein is the true
homologue of the human protein because of two shared characteristics.
Mouse and human K12 bind to CD7 of the homologous species, and both
species of K12 map near the corresponding CD7 locus.
The primary function of the CD7 protein in the immune system is
presently unknown. CD7 is expressed on mature T and NK cells, as well
as on progenitors of T, B, NK, and myeloid cells (9, 10, 14, 15) and on
intestinal intraepithelial lymphocytes (21, 22). CD7 is thought to be a
marker for one of the earliest stages of T cell development (reviewed
in Refs. 23 and 24). Consistent with a role for CD7 in T cell
development is the finding that CD7 was not expressed on T cells from
an infant with severe combined immunodeficiency (25).
Targeted disruption of the CD7 gene in mice has been
achieved by several groups (26, 27). One group found no demonstrable effect on either the function or subsets of lymphocytes, and no effect
on NK cell cytotoxicity (27). However, the second group did note a
transient increase in thymocyte numbers at 3 months, and an alteration
in antigen-specific CTL effector activity (26). These data suggest that
the role of CD7 in healthy animals is subtle at best, and that a better
place to look for a function for CD7 (and K12-Fc as well) might be in
immunologically challenged mice, i.e. those with viral,
bacterial, or parasitic infections. Recently, however, Sempowski and
co-workers (28) demonstrated that CD7-deficient mice were resistant to
lipopolysaccharide-induced shock syndromes, and they suggested that CD7
may be a key molecule in the lipopolysaccharide-induced inflammatory
response. In preliminary experiments, however, we have been unable to
demonstrate either a positive or negative effect of the soluble K12
protein in an lipopolysaccharide/D-galactosamine challenge
model (29) (data not shown). Expression of the human CD7
gene in transgenic mice had no effect on mouse thymopoiesis, even
though the gene was expressed in T cells and was induced during T cell
activation (30). This data is consistent with our finding that mouse
K12-Fc cannot bind to human CD7 (Fig. 5).
Much of what is known about CD7 function comes from studies of the
biological activities of anti-CD7 monoclonal antibodies. These
antibodies have been shown to have a co-stimulatory role, along with
anti-CD3 antibodies, in the activation of T cells (18, 31), and they
can also directly activate Previous efforts to identify a cognate for CD7 have not been
successful, although a putative ligand has reportedly been detected in
serum (38). The extracellular domain of CD7, expressed by either
mammalian or insect cells, has been shown to interact with some
specificity with ConA (38). This interaction is at least partly
mediated through carbohydrate residues on CD7, since treatment of the
extracellular domain of CD7 with glycosidases specifically reduces
binding of ConA to CD7. Recombinant soluble CD7 inhibited antigenic-
and alloantigenic-induced T cell proliferation, suggesting that a cell
bound ligand for CD7 does exist (39). The cross-linking of CD7 by its
putative natural ligand (K12) may be responsible for the costimulatory
role it plays in T cell activation (39).
In their paper describing K12, Slentz-Kesler and co-workers (1)
presented immunofluorescence and flow cytometry data indicating that
K12 was not located on the surface of several breast cancer cell lines
that expressed the protein. This result was surprising given that
hydrophobicity analysis of the K12 protein suggests that it could be a
cell surface protein. We transfected either human or mouse K12 proteins
into COS-1 cells, and then tested their capacity to bind human or mouse
CD7-Fc, respectively (Fig. 6). Strong binding was obtained within
species, indicating that both mouse and human K12 proteins are
expressed on the cell surface, at least on transfected cells, and can
bind CD7 in that location. It may be that in the breast cancer cell
lines examined by Slentz-Kesler and co-workers (1), the K12 protein is
expressed on the cell surface in such small amounts as to preclude
detection. Alternatively, it may be that K12 on the cell surface is
rapidly cleaved to generate a soluble K12 protein. A number of type I
transmembrane ligands undergo proteolytic cleavage on the cell surface
to generate soluble forms, including flt3 ligand, stem cell factor,
colony stimulating factor-1, and epidermal growth factor.
Knowing that K12 binds to CD7 suggests several possible uses for the
K12 protein, or molecules derived from it. CD7 has been suggested to be
involved in both human immunodeficiency virus type 1 infection and
syncytia formation since anti-CD7 antibodies block both of these
processes (36). A soluble form of the K12 protein could therefore
possibly be used to block human immunodeficiency virus infection and
syncytia formation. Since CD7 has been suggested to be a good marker
for T cell leukemias (reviewed in Ref. 40), several groups have created
immunotoxins by fusing anti-human CD7 monoclonal antibodies to toxins
such as ricin (41, 42) or saporin (43). It should be possible to
conjugate toxins to the extracellular domain of K12 as well; these
conjugates may be less immunogenic than antibody-based conjugates, or
may have a longer half-life. Conjugation of the anti-CD7 antibodies
with toxins may not even be required, since anti-CD7 antibodies alone have been effective anti-tumor agents in a xenografted human T cell ALL
model (44). If K12-Fc has a similar effect in this model, it would be a
candidate for clinical testing against T cell leukemias. A monoclonal
antibody directed against CD7 has been shown to inhibit T cell
proliferation in the allogeneic mixed lymphocyte reaction (35). This
study has led to a clinical trial of an anti-CD7 antibody for the
prophylaxis of kidney transplant rejection (45), and again it is
possible that a soluble K12 protein could be used in a similar fashion.
Cross-linking of CD7 via anti-CD7 mAb has been shown to induce
activation of primary human NK cells from peripheral blood (34). This
finding fueled speculation that CD7 may interact with a natural cognate
that could regulate NK cell activation and function. The finding that
K12 can cross-link CD7 and activate human NK cells in a manner similar
to CD7 mAb suggests that epithelial cells or granulocytes (the
predominant cell types that express K12) (1) have the potential to
regulate NK cell activation, cytokine secretion, and function. Since
others have observed that CD7 cross-linking induces biological effects
on T-cells (31, 32), cells that express K12 may also possess the
ability to regulate T-cell function through this cognate interaction.
Our understanding of CD7 biology is heavily skewed at present by the
studies done using antibodies directed against this protein. It will be
interesting to determine if the activities of the antibodies are the
same or different from those activities of soluble K12 protein. Future
experiments should help to delineate the range of interactions between
CD7 and K12, as well as the biological processes that are effected
by these interactions.
We thank Lisa Parshley for generating the
phytohemaglutinin stimulated human PBT cDNA expression library, and
Chang-Pin Huang for sequencing the CD7 clones in the
expression screen, Ilka Havukkala of Genesis Research and Development
(Auckland, New Zealand) for supplying the 971012tram001354ht sequence,
Della Friend for performing the preliminary Scatchard analysis of
K12-Fc binding, and Jo Viney and Gina Westrich for the chromosomal
mapping of mouse K12. We also thank Adel Youakim and Peter Baum for
numerous helpful discussions, Gary Carlton for help with preparation of
the figures and electronic submission, and Mike Widmer and Doug
Williams for reviewing the manuscript.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF210700.
The abbreviations used are:
PCR, polymerase
chain reaction;
PBS, phosphate-buffered saline;
FBS, fetal bovine
serum;
ConA, concanavalin A;
FACS, fluorescence cell sorter activator;
mAb, monoclonal antibody;
PE, phosphatidylethanolamine.
Identification of CD7 as a Cognate of the Human K12 (SECTM1)
Protein*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
induced proliferation, of mouse lymph node T cells. Human K12-Fc stimulated the up-regulation of CD25,
CD54, and CD69 on human NK cells in vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Zap library. All cDNA clones were sequenced on both strands to
confirm no amino acid changes had been introduced by PCR into the
published CD7 sequence (9, 10).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Flow cytometric analysis of human K12-Fc
fusion protein binding to control (panel A) or
ConA-stimulated (panel B) e-rosetted human T
cells.
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Fig. 2.
Human K12-Fc precipitates an approximate
40-kDa protein from primary human NK cells. NK cells were
isolated, radiolabeled with [35S]cysteine/methionine,
lysed, and incubated with K12-Fc fusion as described under
"Experimental Procedures." The precipitated proteins were separated
by SDS-PAGE, the gel was dried, and the proteins were visualized by
autoradiography.
Binding of a panel of anti-human CD7 monoclonal antibodies to Jurkat
cells (a human T cell leukemia cell line), and the effect of K12-Fc
on that binding
View larger version (45K):
[in a new window]
Fig. 3.
Sequence comparison of the human K12 protein
and its putative mouse homologue. Protein sequences were compared
using the GCG program GAP. The arrow indicates the predicted
signal peptide cleavage site in both the human and mouse proteins.
Potential N-linked glycosylation sites are
underlined, and the core transmembrane regions (predicted by
the TRANSMEMBRANE program) as well as conserved cysteine residues are
boxed.
View larger version (20K):
[in a new window]
Fig. 4.
Flow cytometric analysis of mouse K12-Fc
fusion protein binding to control (panel A) or
ConA-stimulated (panel B) mouse lymph node
cells.
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[in a new window]
Fig. 5.
Binding of human and mouse K12-Fc fusion
proteins to COS-1 cells that have been transfected with cDNAs
encoding human or mouse CD7. Binding assays were done as described
under "Experimental Procedures," and the bound K12-Fc proteins were
quantified on a PhosphorImager.
1 on both cell types (data not shown).
View larger version (11K):
[in a new window]
Fig. 6.
Binding of mouse and human CD7-Fc fusion
proteins to cells transfected with cDNAs encoding the transmembrane
forms of either mouse or human K12 proteins. The binding assays
were done as described under "Experimental Procedures."
/ (Fig. 7B). The K12-Fc fusion protein inhibited
ConA-induced proliferation of the cells in a dose-dependent
manner, but had no effect on anti-TcR-induced cell proliferation. Since
ConA is known to bind to a number of proteins, including CD7, the
inhibition of ConA-induced T cell proliferation by K12-Fc could simply
be due to K12 blocking of ConA binding to CD7. We therefore examined whether ConA could inhibit K12-Fc binding to COS-1 cells transfected with cDNAs encoding CD7. No blocking of either human K12-Fc (added at a concentration of 1 µg/ml) to human CD7 (Fig.
8A) or mouse K12-Fc (added at
a concentration of 1 µg/ml) to mouse CD7 (Fig. 8B) was
seen when the cells were preincubated with 20 µg/ml ConA. When the
amount of ConA in the medium was raised to 2 mg/ml, human K12-Fc
binding was inhibited approximately 30% (Fig. 8A) and mouse K12-Fc binding was inhibited about 90% (Fig. 8B). Given
that the concentration of ConA used in the cell proliferation
experiment was 1 µg/ml, it seems unlikely that the inhibitory effect
of K12-Fc on the proliferation of lymph node cells was due to the
blocking of ConA binding to the cells.
View larger version (18K):
[in a new window]
Fig. 7.
Mouse K12-Fc inhibits ConA induced
proliferation of mouse lymph node cells in a dose-dependent
manner (panel A), but does not block the anti-TcR
/-induced proliferation of
mouse lymph node cells (panel B).
View larger version (13K):
[in a new window]
Fig. 8.
The effect of ConA on the binding of human
K12-Fc to human CD7 (panel A) or mouse K12-Fc to mouse
CD7 (panel B). The cells were transfected with
the indicated CD7 cDNAs (or vector only, designated pDC409),
preincubated with the indicated concentrations of ConA, and then
incubated with the indicated K12-Fc fusion protein (each at a
concentration of 1 µg/ml) as described under "Experimental
Procedures."
Effect of CD7 cross-linking on human NK cell activation as determined
by flow cytometry
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
T cells (32). Antibody-induced
ligation of CD7 on the surface of either T cells or NK cells leads to
the phosphorylation of intracellular proteins in those cells (20).
Cross-linking of CD7 on T cells increases adhesion of the cells to
fibronectin, ICAM-1, and V-CAM1 (33), and cross-linking of CD7 on NK
cells has been shown to induce adhesion to fibronectin (34). Triggering
CD7 with antibodies has also been shown to regulate the functional
activity of 1 integrins on NK cells (34). Antibodies to
CD7 inhibit proliferation in allogeneic and autologous mixed lymphocyte
reactions (35). Anti-CD7 antibodies stimulate NK cell proliferation and
enhance the cytotoxicity of the cells, in addition to inducing
interferon production (34). Studies with anti-CD7 antibodies
suggest that CD7 may function as an accessory protein in human
immunodeficiency virus type 1 mediated syncytium formation as well as
infection (36). The effects of anti-CD7 antibodies are not limited to T
and NK cells, as they also stimulate granulocyte macrophage-colony stimulating factor production by several myeloid cell lines (37).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 206-389-4329;
Fax: 206-682-9927; E-mail: slyman@immunex.com.
ABBREVIATIONS
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INTRODUCTION
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DISCUSSION
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