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. TheK12 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 on35S-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 mouseCD7 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-TcRα/β 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. 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. TheK12 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 on35S-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 mouseCD7 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-TcRα/β 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. polymerase chain reaction phosphate-buffered saline fetal bovine serum concanavalin A fluorescence cell sorter activator monoclonal antibody phosphatidylethanolamine The K12 (SECTM1) gene was originally identified (1.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar) as being directly 5′ of the locus encoding the humanCD7 gene on human chromosome 17 (2.Osada S. Utsumi K.R. Ueda R. Akao Y. Tsuge I. Nishida K. Okada J. Matsuoka H. Takahashi T. Cytogenet. Cell Genet. 1988; 47: 8-10Crossref PubMed Scopus (9) Google Scholar). The 3′ end of theK12 gene is about 5 kilobases upstream of the start of the human CD7 gene; both genes are transcribed in the same direction (1.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar). The human K12 protein has been shown to be primarily expressed in spleen, prostate, testis, small intestine, and in peripheral blood leukocytes (1.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar). 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.Lyman S.D. James L. Vanden Bos T. de Vries P. Brasel K. Gliniak B. Hollingsworth L.T. Picha K.S. McKenna H.J. Splett R.R. Fletcher F.F. Maraskovsky E. Farrah T. Foxworthe D. Williams D.E. Beckmann M.P. Cell. 1993; 75: 1157-1167Abstract Full Text PDF PubMed Scopus (469) Google Scholar, 4.Hannum C. Culpepper J. Campbell D. McClanahan T. Zurawski S. Bazan J.F. Kastelein R. Hudak S. Wagner J. Mattson J. Luh J. Duda G. Martina N. Peterson D. Menon S. Shanafelt A. Muench M. Kelner G. Namikawa R. Rennick D. Roncarolo M.-G. Zlotnick A. Rosnet O. Dubreuil P. Birnbaum D. Lee F. Nature. 1994; 368: 643-648Crossref PubMed Scopus (393) Google Scholar), c-kit ligand (5.Anderson D.M. Lyman S.D. Baird A. Wignall J.M. Eisenman J. Rauch C. March C.J. Boswell H.S. Gimpel S.D. Cosman D. Williams D.E. Cell. 1990; 63: 235-243Abstract Full Text PDF PubMed Scopus (727) Google Scholar, 6.Martin F.H. Suggs S.V. Langley K.E. Lu H.S. Ting J. Okino K.H. Morris C.F. McNiece I.K. Jacobsen F.W. Mendiaz E.A. Birkett N.C. Smith K.A. Johnson M.J. Parker V.P. Flores J.C. Patel A.C. Fisher E.F. Erjavec H.O. Herrera C.J. Wypych J. Sachdev R.K. Pope J.A. Leslie I. Wen D. Lin C. Cupples R.L. Zsebo K.M. Cell. 1990; 63: 203-211Abstract Full Text PDF PubMed Scopus (602) Google Scholar, 7.Huang E. Nocka K. Beier D.R. Chu T.-Y. Buck J. Lahm H.W. Wellner D. Leder P. Besmer P. Cell. 1990; 63: 225-233Abstract Full Text PDF PubMed Scopus (942) Google Scholar), and colony stimulating factor 1 (8.Cerretti D.P. Wignall J. Anderson D. Tushinski R.J. Gallis B.M. Stya M. Gillis S. Urdal D.L. Cosman D. Mol. Immunol. 1988; 25: 761-770Crossref PubMed Scopus (109) Google Scholar). 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 mouseCD7 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. The human K12 protein was cloned based on the published sequence (1.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar) using reverse transcriptase-PCR1from 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 λ 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.Yoshikawa K. Seto M. Ueda R. Obata Y. Fukatsu H. Segawa A. Takahashi T. Immunogenetics. 1993; 37: 114-119Crossref PubMed Scopus (16) Google Scholar, 10.Yoshikawa K. Seto M. Ueda R. Obata Y. Aoki S. Takahashi T. Immunogenetics. 1995; 41: 159-161PubMed Google Scholar). 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.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar), 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). The human peripheral blood T cell library cDNA expression library was constructed in the pDC409 vector using methods previously described (11.McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan C.I. Copeland N.G. Huebner K. Croce C.M. Cannizzarro L.A. Benjamin D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (622) Google Scholar) 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.Lyman S.D. James L. Vanden Bos T. de Vries P. Brasel K. Gliniak B. Hollingsworth L.T. Picha K.S. McKenna H.J. Splett R.R. Fletcher F.F. Maraskovsky E. Farrah T. Foxworthe D. Williams D.E. Beckmann M.P. Cell. 1993; 75: 1157-1167Abstract Full Text PDF PubMed Scopus (469) Google Scholar). 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. 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. 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.Lyman S.D. James L. Vanden Bos T. de Vries P. Brasel K. Gliniak B. Hollingsworth L.T. Picha K.S. McKenna H.J. Splett R.R. Fletcher F.F. Maraskovsky E. Farrah T. Foxworthe D. Williams D.E. Beckmann M.P. Cell. 1993; 75: 1157-1167Abstract Full Text PDF PubMed Scopus (469) Google Scholar) with the following modifications. The binding media (RPMI 1640, 1% FBS, 0.02% sodium azide, 20 mmHEPES, pH 7.2) was modified to include 1 mmMnCl2. 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.Lyman S.D. James L. Vanden Bos T. de Vries P. Brasel K. Gliniak B. Hollingsworth L.T. Picha K.S. McKenna H.J. Splett R.R. Fletcher F.F. Maraskovsky E. Farrah T. Foxworthe D. Williams D.E. Beckmann M.P. Cell. 1993; 75: 1157-1167Abstract Full Text PDF PubMed Scopus (469) Google Scholar). After binding the iodinated antibody, the cells were washed and then the radioactivity quantified by Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The chromosomal location of the putative mouse K12 protein was determined using the Jackson Laboratory radiation hybrid panel mapping resource (12.Flaherty L. Herron B. Mamm. Genome. 1998; 9: 417-418Crossref PubMed Scopus (20) Google Scholar). 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 nml-arginine, 272 nml-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+). 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 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). 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 CD7gene. The size of the protein precipitated from NK cell lysates (40 kDa) is consistent with the reported size of human CD7 (13.Haynes B.F. Immunol. Rev. 1981; 57: 127-161Crossref PubMed Scopus (137) Google Scholar,14.Aruffo A. Seed B. EMBO J. 1987; 6: 3313-3316Crossref PubMed Scopus (109) Google Scholar). 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 (TableI).Table IBinding 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 bindingSection 1NonblockedPreblocked with HuK12FCPositive cellsMFIPositive cellsMFIPrimary antibodyAnti-CD7 clone M-T70178%2712%19Anti-CD7 clone 8118.178%278%24Anti-CD7 clone 4H988%384%19Anti-CD7 clone RFT-2a71%239%18Anti-CD7 clone WM3162%2211%18Anti-CD7 clone CLB-3A180%3063%24IgG isotype control3%18Section 2NonblockedPreblocked with various anti-CD7 AbsPositive cellsMFIPositive cellsMFIPrimary proteinHuK12-Fc98%956%16 - (clone M-T701)62%23 - (clone 8118.1)85%30 - (clone 4H9)80%27 - (clone RFT-2a)12%17 - (clone WM31)73%27 - (clone CLB-3A1)97%94 - IgG isotype controlControl Fc protein3.4%18IgG control3.6%17Section 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. Open table in a new tab 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. 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.Yoshikawa K. Seto M. Ueda R. Obata Y. Notake K. Yokochi T. Takahashi T. Immunogenetics. 1991; 33: 352-360Crossref PubMed Scopus (6) Google Scholar), 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.Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (400) Google Scholar). 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 Aand B). The humanK12 gene is adjacent to the human CD7 gene on human chromosome 17 (1.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar). The chromosomal location of the putative mouseK12 gene was determined by radiation hybrid mapping (12.Flaherty L. Herron B. Mamm. Genome. 1998; 9: 417-418Crossref PubMed Scopus (20) Google Scholar). The mouse K12 gene is localized just proximal to theCD7 locus on mouse chromosome 11 (17.Lee D.M. Watson M.L. Seldin M.F. Immunogenetics. 1994; 39: 289-290Crossref PubMed Scopus (5) Google Scholar) 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. 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 mouseCD7 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 × 108m−1 on both cell types (data not shown). Slentz-Kesler and co-workers (1.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar) 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 mouseK12 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.Slentz-Kesler K.A. Hale L.P. Kaufman R.E. Genomics. 1998; 47: 327-340Crossref PubMed Scopus (24) Google Scholar) 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). 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.7 A) or immobilized anti-TcR α/β (Fig. 7 B). 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.8 A) or mouse K12-Fc (added at a concentration of 1 μg/ml) to mouse CD7 (Fig. 8 B) 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. 8 A) and mouse K12-Fc binding was inhibited about 90% (Fig. 8 B). 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.Figure 8The 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."View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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).Table IIEffect of CD7 cross-linking on human NK cell activation as determined by flow cytometryCulture conditionaNK 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.Control mAbCD25bResults are expressed as the mean fluorescence intensity of binding for the PE-conjugated antibody listed (CD25, CD69, or CD54).CD69CD54Control IgG (2.5 μg/ml)271512huCD7 mAb (2.5 μg/ml)5161843huK12-Fc (2.5 μg/ml)7385762a 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 do