Identification of CD7 as a cognate of the human K12 (SECTM1) protein.

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 IgG(1). 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 (35)S-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-TcRalpha/beta 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.

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.

EXPERIMENTAL PROCEDURES
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-PCR 1 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 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).
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 ϫ 10 6 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 ϫ 10 6 cells/ml) were radiolabeled overnight with 50 Ci/ml [ 35 S]cysteine/methionine) (ProMix, Amersham Pharmacia Biotech). Radiolabeled cells were lysed with 1 ml of RIPA E lysis buffer (PBS, 1% Triton). 150 l of lysate were incubated with 1 g 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, * 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF210700.
K12/CD7 Binding Studies-COS-1 cells were transfected with fulllength 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 MnCl 2 . In some experiments, the transfected cells were incubated with zero, 20 g/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 g/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 125 I-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.
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 l of streptavidin/Dynabeads (Dynal, Oslo, Norway) per 10 ϫ 10 6 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 ϩ , CD16 ϩ ).
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 g/ml in PBS. The human NK cells were added at 2 ϫ 10 6 /ml in RMPI containing 5% FBS and incubated for 20 h at 37°C in 5% CO 2 .
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 ϫ 10 6 cells was incubated with the indicated mAb-fluorochrome conjugates for 1 h at 4°C in a total volume of 100 l. The cells were then washed in 2 ml of PBS containing 2% FBS and 0.1% azide and resuspended in 300 l 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).

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 [ 35 S]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

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.
CD7 Is a Cognate of the Human K12 (SECTM1) Protein 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 212amino 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 (K a ) of human K12-Fc for human CD7 was estimated to be in the range of 1 ϫ 10 8 M Ϫ1 on both cell types (data not shown).
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 IgG 1 ) 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 ␣/␤ (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 g/ml) to human CD7 (Fig. 8A) or mouse K12-Fc (added at a concentration of 1 g/ml) to mouse CD7 (Fig. 8B) was seen when the cells were preincubated with 20 g/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 g/ml, it CD7 Is a Cognate of the Human K12 (SECTM1) Protein 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.
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 -6fold 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 crosslinking 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).  T cell leukemia cell line), and the effect of K12-Fc on that binding Section 1 shows the reductions in the percentage of positive cells and mean fluorescence intensity that occur when Jurkat cells are preincubated with human K12-Fc fusion protein prior to staining the cells with the antibodies. Section 2 shows that, in a similar fashion, preincubation of the Jurkat cells with the six anti-human CD7 antibodies blocks the binding of human K12-Fc to the cells.  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.

DISCUSSION
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 lympho- cytes, 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 coworkers (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 ␥/␦ 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).
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 alloantigenicinduced 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 delin- a NK cells were cultured in the presence of plate-bound molecules listed under culture condition; the huCD7 mAb was clone 8118.1. Results are from one of four representative experiments performed using NK cells from a single donor.
b Results are expressed as the mean fluorescence intensity of binding for the PE-conjugated antibody listed (CD25, CD69, or CD54). eate the range of interactions between CD7 and K12, as well as the biological processes that are effected by these interactions.