CD96 Interaction with CD155 via Its First Ig-like Domain Is Modulated by Alternative Splicing or Mutations in Distal Ig-like Domains
2008; Elsevier BV; Volume: 284; Issue: 4 Linguagem: Inglês
10.1074/jbc.m807698200
ISSN1083-351X
AutoresDorothee Meyer, Sebastian Seth, Jana D. Albrecht, Michael K. Maier, Louis Du Pasquier, Inga Ravens, Lutz Dreyer, Renate Burger, Martin Gramatzki, Reinhard Schwinzer, Elisabeth Kremmer, Reinhold Foerster, Günter Bernhardt,
Tópico(s)T-cell and B-cell Immunology
ResumoThe adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes. The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes. CD96 is a single pass transmembrane glycoprotein belonging to the Ig superfamily (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). In its extracellular part, CD96 consists of three Ig-like domains in the order VN-term-V-CC-term and a membrane proximal region rich in proline/serine/threonine. It is assumed that extensive O-linked glycomodification of this region equips CD96 with a semi-flexible stalk. Therefore, the Ig-like domains may protrude among other membrane-bound macromolecules comprising the outer layer of the cell (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). Even though such exposed presentation predestines CD96 for roles in adhesion, interaction partners of CD96 as well as its biological function(s) remained largely obscure until recently. However, CD96 was described as tumor marker for T-ALL and AML characteristic for the AML stem cell in particular (2Gramatzki M. Ludwig W.D. Burger R. Moos P. Rohwer P. Grunert C. Sendler A. Kalden J.R. Andreesen R. Henschke F. Moldenhauer G. Exp. Hematol.. 1998; 26: 1209-1214Google Scholar, 3Hosen N. Park C.Y. Tatsumi N. Oji Y. Sugiyama H. Gramatzki M. Krensky A.M. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 11008-11013Google Scholar). Among human peripheral blood cells, CD96 expression was observed on T and NK cells but not on the majority of B cells, monocytes, and granulocytes (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar). The murine CD96, characterized recently, is widely expressed on T cells of all developmental and differentiation stages tested albeit to varying extents (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). This is in line with the original observation of elevated CD96 expression in activated human T cells (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). In humans, CD96 promotes adhesion of NK cells to tumor cells expressing CD155, an interaction that may assist in NK-mediated destruction of the targets (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar); yet direct evidence proving CD96 as NK receptor is missing. Although CD155 remained the only identified interaction partner for CD96 in human (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar), nectin-1 also binds to CD96 in mouse (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). Although the significance of the CD96/nectin-1 interaction remains unclear, it is interesting to note that nectin-1 is expressed prominently in brain and, in particular, localizes to the synaptic junctions. Nectin-1-deficient mice suffer from microphthalmia (small eyes), an eye disorder caused by malformation of cell layers of the ciliary epithelia (6Inagaki M. Irie K. Ishizaki H. Tanaka-Okamoto M. Morimoto K. Inoue E. Ohtsuka T. Miyoshi J. Takai Y. Development.. 2005; 132: 1525-1537Google Scholar). In humans, mutations in nectin-1 were associated with the occurrence of cleft lip/palate-ectodermal dysplasia syndrome (7Suzuki K. Hu D. Bustos T. Zlotogora J. Richieri-Costa A. Helms J.A. Spritz R.A. Nat. Genet.. 2000; 25: 427-430Google Scholar), whereas a mutation in CD96 (T280M) or disruption of the genetic locus of CD96 by chromosomal translocation link to a distinct manifestation of the C syndrome (Opitz Trigonocephaly) (8Kaname T. Yanagi K. Chinen Y. Makita Y. Okamoto N. Maehara H. Owan I. Kanaya F. Kubota Y. Oike Y. Yamamoto T. Kurosawa K. Fukushima Y. Bohring A. Opitz J.M. Yoshiura K. Niikawa N. Naritomi K. Am. J. Hum. Genet.. 2007; 81: 835-841Google Scholar). Nectin-1 as well as CD155 belong to a subfamily of Ig-like adhesive receptors (9Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci.. 2003; 94: 655-667Google Scholar). An evolutionary analysis revealed that the members of this family share a common progenitor with CD96 and CD226 (10Du Pasquier L. Zucchetti I. De Santis R. Immunol. Rev.. 2004; 198: 233-248Google Scholar), another binding partner for CD155 (11Bottino C. Castriconi R. Pende D. Rivera P. Nanni M. Carnemolla B. Cantoni C. Grassi J. Marcenaro S. Reymond N. Vitale M. Moretta L. Lopez M. Moretta A. J. Exp. Med.. 2003; 198: 557-567Google Scholar). Mice deficient for CD155 produce a reduced humoral immune response to soluble antigens entering the organism via the gastrointestinal tract, whereas subcutaneous or systemic immunizations are not affected (12Maier M.K. Seth S. Czeloth N. Qiu Q. Ravens I. Kremmer E. Ebel M. Muller W. Pabst O. Forster R. Bernhardt G. Eur. J. Immunol.. 2007; 37: 2214-2225Google Scholar). These findings document the involvement of CD96 and members of the CD155/nectin family in immunological as well as developmental processes. In this study we were interested to investigate the interaction of CD96 with its binding partners in more detail. To this end, chimeric receptors were generated consisting of distinct mouse and human Ig-like domains. It could be demonstrated that the first domain of CD96 harbors the epitope(s) essentially required for CD155 binding. However, the magnitude of binding is modulated by the second domain in case of human but not mouse CD96. Contrary to murine CD96, human CD96 is alternatively spliced, giving rise to two isoforms differing in the Ig fold of the second domain. Expression analysis by real time PCR revealed that the shorter variant is predominantly expressed in all nonmalignant tissue and cell types investigated. CD155 binds to both splice variants yet with different strength. Moreover, the T280M mutation residing in Ig-like domain three of CD96 resulted in a significantly reduced binding to CD155. Cloning of Human CD96 cDNA—Full-length hCD96 4The abbreviations used are: hCD, human CD; mCD, murine CD; mAb, monoclonal antibody; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; EYFP, enhanced yellow fluorescent protein; T-ALL, T-cell acute lymphoblastic leukemia; AML, acute myeloid leukemia; PBSd, PBS deficient in Ca2+ and Mg2+. was cloned out of white blood cell cDNA (Clontech) by PCR (Expand high fidelity; Roche Applied Science). The cycling conditions were: 1 min at 94 °C, then 10 cycles consisting of 15 s at 94 °C, 30 s at 55 °C, 2 min at 72 °C, and an additional 15 cycles of 15 s at 94 °C, 30 s at 55 °C, 2 min at 72 °C with an extension time of 5 s/cycle. The primers used were: HCD96.KPN, 5′-GCG GTA CCG GTG TTC AGA AGA CAA TGG AG-3′; and hcd96_xba, 5′-GCT CTA GAC TAG AGG GTC TCC ATC TCA TGA T-3′. After cloning into pcDNA3, the products were sequenced. Two types of cDNA inserts were obtained (see also Ensembl data base entry ENSG00000153283). Variant 1 (hCD96V1) represents the hCD96 version carrying exon 4, whereas in variant 2 (hCD96V2) this exon is missing, causing the absence of 18 amino acids. Exon 4 borders are in frame (phase 0) such that differential splicing does not cause additional amino acid changes. Detection of hCD96 Proteins by Western Blotting—To visualize hCD96 proteins, the pCDNA3 plasmids driving the expression of murine ICAM-1, hCD96V1 or hCD96V2 were transiently transfected into HEK293 cells (see below). After 2 days the cells were surface-biotinylated. The AML cell line KG1 endogenously expressing hCD96 served as a positive control. For biotinylation, ∼5 × 107 cells were detached with PBSd, 10 mm glucose/2 mm EDTA (only for HEK293) and washed twice with PBSd, 10 mm glucose and resuspended in 1 ml of PBSd, 10 mm glucose. 2 mg of biotinylation reagent (No-weigh NHS-PEO4-Biotin; Thermo Scientific) was dissolved in 1 ml of PBSd, 10 mm glucose and added to the cells. The mixture was slowly rotated at room temperature for 30 min and then washed twice with 15 ml of PBSd, 10 mm glucose. The pellet was resuspended in 1 ml of PBSd, 0.25% Nonidet P-40, incubated for 20 min on ice, and centrifuged to obtain cell extract. For immunoprecipitations, the cell extracts were rocked gently either with 10 μg of anti-hCD96 antibody TH-111 (2Gramatzki M. Ludwig W.D. Burger R. Moos P. Rohwer P. Grunert C. Sendler A. Kalden J.R. Andreesen R. Henschke F. Moldenhauer G. Exp. Hematol.. 1998; 26: 1209-1214Google Scholar) or 20 μg of recombinant hCD155-hIgG protein along with 25 μl of protein G-Sepharose beads (GE Healthcare) overnight at 4 °C. The beads were then washed twice with PBSd, 0.25% Nonidet P-40 and twice with PBSd. The beads were boiled in 30 μl of Laemmli buffer. Following SDS-PAGE on a 10% BisTris gel (Invitrogen), the proteins were blotted, and the biotinylated material was revealed after blocking with dry milk in TBST and incubation/detection with streptavidin-alkaline phosphatase (Sigma) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Construction of Chimeric and Mutant Receptors—Full-length cDNAs coding for hCD96V2 and mCD96 were subjected to site-directed mutagenesis to create ClaI or BlpI sites according to the manufacturer's instruction (Stratagene). Although the introduction of the BlpI site was silent, that of the ClaI site caused one amino acid substitution (hCD96, A142T; mCD96, Q141T). Domain interchanges were done applying appropriate restriction enzyme digests. The T280M mutation was inserted into both hCD96 cDNA versions. All of the mutant clones were sequenced to verify their integrity. The primers used were: HCD96D12, 5′-CAT TCA GAC ACA CGT TAC AAT CGA TGA ATG GAA CAG CAA CC; hcd96_d12 5′-GGT TGC TGT TCC ATT CAT CGA TTG TAA CGT GTG TCT GAA TG; HMCD96D23, 5′-TCC ACC ACA GTC AAG GTT TTT GCT AAG CCA GAA ATC C; hmcd96_d23, 5′-GGA TTT CTG GCT TAG CAA AAA CCT TGA CTG TGG TGG A; MUCD96D12, 5′-CAT TGT GGA ACC CTA TAC AAT CGA TGA ACA CAA CTA TAC AAT AG; mucd96_d12, 5′-CTA TTG TAT AGT TGT GTT CAT CGA TTG TAT AGG GTT CCA CAA TG; CD96T280M, 5′-GTG ATT GTG GAA AAT AAC TCC ATG GAT GTC TTG GTA G; and cd96_t280m, 5′-CTA CCA AGA CAT CCA TGG AGT TAT TTT CCA CAA TCA C. Transient Transfection and Binding of Antibodies or Recombinant Proteins—Twenty μg of cDNA encoding the diverse human/murine CD96 versions were transfected into HEK293 cells along with 2 μg of a green fluorescent protein-expressing plasmid applying the standard calcium phosphate transfection technique. The cells were detached 48 h later and incubated with either anti-hCD96 antibody TH-111 or a panel of anti-mCD96 antibodies clones 6A6, 8B10, or 6B4, respectively (see Ref. 5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar for details). Rat anti-mouse IgG-Cy5 or mouse anti-rat IgG-Cy5 secondary antibodies were used for detection. CD155 and nectin-1 were expressed and purified as recombinant hIgG1 proteins as described earlier (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). They consist of the entire ectodomain of CD155 or nectin fused to domains 3 + 4 of hIgG1. Transfected cells were first incubated with 2 μg of recombinant protein on ice and washed twice, and bound protein was detected by mouse anti-human biotin/streptavidin PerCP. For titration of recombinant protein on HEK293 cells transiently expressing hCD96V1, hCD96V1m, hCD96V2, or hCD96V2m, respectively, the cells were incubated with the indicated amounts of recombinant hCD155-hIgG1 in 50 μl of PBSd, 2% fetal calf serum for 1 h on ice. After three washes bound protein was detected using goat anti-hIgG-bio/streptavidin Cy5. In parallel samples, the cells were incubated with decreasing amounts of mAb TH-111 and washed, and bound mAb revealed by adding rat anti-mouse IgG-Cy5. For flow cytometry, propidium iodide was added to exclude dead cells from analysis. The cells were analyzed on a FACSCalibur or LSRII (BD Biosciences), and the data were evaluated using WinList5.0. The binding of mAb and recombinant protein (as seen in Fig. 3) was normalized to cover a range from 0 to 100, where 100 was assigned to the binding to the parental receptors (hhh in case of TH-111 and hCD155-IgG; mmm for 6A6, 8B10, 6B4, and mCD155-IgG, respectively) using the following formula. relative binding=linMeanX−linMeanXbase linelinMeanXphysiological−linMeanXbase line⋅100(Eq. 1) Three independent experiments were included in the analysis, allowing the calculation of the mean and S.D. for representation. The TH-111 binding efficiencies to different hCD96 variants (Fig. 4) were determined by titration. The linMeanX values were normalized for plotting using the following formula. normalized mAb binding=linMeanXsample−linMeanXbase linelinMeanXmax−linMeanXbase line⋅100(Eq. 2) Signals detected from protein binding were corrected to include the expression level of each CD96 variant by dividing by the half-maximal signal of mAb TH-111. corrected binding=linMeanX(CD155)linMeanX50(TH−111)(Eq. 3) The results were then normalized to a scale from 0 to 100, thereby adjusting the strongest binding (elicited by hCD96V2 binding to the highest amount of hCD155-IgG) to 100. Three independent experiments were included in the analysis, allowing the calculation of the mean and S.D. for representation. Electroporation of Raji Cells and Cell Adhesion Assay—Raji cells were grown in RPMI 1640, 10% fetal calf serum and taken up at 2 × 107 cells/ml in growth medium following centrifugation. 400 μl of cells were mixed in a cuvette (0.4 cm electrode gap) with 10–20 μg of expression plasmid, 10 μl of 0.2 m ATP, and 3 μl of 1 m MgCl2. Electroporation was done at 240 V and 1,500 microfarad. The cells were then kept for 2 days in growth medium. For cell adhesion assays, EYFP-tagged hCD96 receptors were constructed. First, the XbaI fragment of vector pEYFP (Clontech) containing the open reading frame for the fluorescent protein EYFP was cloned into pCDNA3 (Invitrogen) to yield vector pCDNA3-EYFP. Next, hCD96 fusion fragments encompassing the entire ectodomain of either hCD96 variant were generated by PCR for in frame cloning into the BamHI site of pCDNA3-EYFP. PCR conditions were as described above with the full-length hCD96 clones as templates using the following primers: HSCD96.BAM, 5′-GCG GAT CCG GTG TTC AGA AGA CAA TGG AG; and hscd96_bamfus, 5′-GCG GAT CCA GGG TCT CCA TCT CAT GAT AAG G. The correct sequences of the fusion clones, hCD96V1-EYFP and hCD96V2-EYFP, was verified by sequencing. Expression of the fusion proteins following transfection into HEK293 cells confirmed that EYFP fluorescence linearly increased with the amount of bound anti-hCD96 antibody, thus providing a tool for monitoring expression efficiency (not shown). 96-well plates were coated with 1 μg/100 μl of PBSd/well of recombinant hCD155-hIgG or murine nectin-2-hIgG protein overnight at 4 °C. The wells were washed with RPMI 1640, 10% fetal calf serum before 2 × 106 Raji cells in ∼200 μl of growth medium/well, transiently expressing equal percentages, and amounts of hCD96 fusion receptors were seeded into 96-well plates in triplicate. Raji cells expressing cytosolic EYFP following transfection of vector pCDNA3-EYFP served as controls. The plates were then spun shortly and incubated for 90 min. at 37 °C. Unbound cells were then removed by two washing steps using 200 μl of growth medium. The attached cells were quantified using the CellTiter96 nonradioactive cell proliferation assay (Promega) according to the instructions of the manufacturer. Isolation of Primary Human Cells, RNA Preparation, and Real Time PCR—White blood cells from healthy donors were prepared using a Biocoll (Biochrom) gradient. The obtained cells from 40 ml of blood were MACS sorted using the CD56 MultiSort kit (Miltenyi), and the CD56+ cell population was stained with mouse anti-hCD3-PerCP (BD Biosciences) to sort out CD3- NK cells on a FACSAria (BD Biosciences). The CD3+ CD4+ and CD3+ CD8+ subpopulations were isolated accordingly from human blood following MACS separation of CD3+ cells with CD3 MicroBeads (Miltenyi) and subsequent staining with anti-CD4/anti-CD8 mAb (in-house preparations) and flow cytometric separation. Monocytes and B cells were obtained as CD14+ and CD20+ fractions following MACS separation. The purity (>95%) of all isolated populations was confirmed by reanalysis. Total RNA was prepared using the Absolutely RNA Microprep Kit (Stratagene). RNA was reverse transcribed (Superscript II reverse transcriptase; Invitrogen) using random hexamer primers. For other human fetal and adult tissues, premade template cDNAs were used (human MTC panel II, human immune system MTC panel, and human fetal MTC panel; Clontech). The ages of the spontaneously aborted fetuses were: 20–33 weeks (fetal brain), 16–32 weeks (fetal spleen), 21–37 weeks (fetal kidney), and 16–32 weeks (fetal thymus). cDNA samples representing AML and T-ALL (13Cario G. Izraeli S. Teichert A. Rhein P. Skokowa J. Moricke A. Zimmermann M. Schrauder A. Karawajew L. Ludwig W.D. Welte K. Schunemann H.J. Schlegelberger B. Schrappe M. Stanulla M. J. Clin. Oncol.. 2007; 25: 4813-4820Google Scholar) were obtained from Dr. J. Krauter (Hannover Medical School) and Dr. M. Stanulla (Hannover Medical School). The expression of hypoxanthine phosphoribosyl transferase, hCD96V1, and hCD96V2 was analyzed using a Lightcycler 2.0 (Roche Applied Science) and the Fast Start DNA Master plus SYBR Green Kit (Roche Applied Science) or the Sybr Premix Ex Taq Kit (Takara). Standardization and absolute relative quantification of expression levels was done as described (14Czeloth N. Bernhardt G. Hofmann F. Genth H. Forster R. J. Immunol.. 2005; 175: 2960-2967Google Scholar). hCD96 primer combinations used were RTPC96UP/rtpc96_ex4 to detect hCD96V1 and RTPC96UP/rtpc96_alt35 for hCD96V2. Both PCR products were cloned, and the inserts were verified by sequencing. These plasmids were then applied as internal standards and also used to demonstrate that the hCD96V1 specifying primer set did not yield any signal on the hCD96V2 template and vice versa. The primers used were: RTPCD96UP, 5′-AAC AGC AAC CAT ACG ATA GAA ATA GA; rtpcd96_alt35, 5′-TTC CAT TAT CCT CCA CCG ACC; and rtpcd96_ex4, 5′-CTT AGA AAG AAG GAC CCA AGA ATC. Ig Folding Prediction and β-Strand Assignment—The sequences representing the Ig-like domains of CD96 were sent to the PHYRE server of the Imperial College (London, UK) and to the Jpred server of the Barton group, University of Dundee for secondary structure predictions. Human CD96 Exists in Two Splice Variants Affecting the Ig Fold of the Second Domain—When cloning human CD96, we noticed a PCR product that represented a version of CD96 lacking exon 4 (Fig. 1, A and D). This exon is 48 nucleotides in length, preserves the open reading frame when spliced out, and maps to the second Ig-like domain (Fig. 1, A and B). A closer analysis involving strand prediction using two different programs suggests that this Ig-like domain belongs to the V-like subset when containing exon 4 amino acids (hCD96V1), whereas the domain encoded by mRNA lacking exon 4 adopts features resembling an I- or a C-like domain, despite maintaining a V-like core folding pattern (hCD96V2). Notably, we failed to identify the corresponding splice variants in mouse. Alignments suggest that murine CD96 is equivalent to human variant2(i.e. the short one). Attempts to detect a putative variant 1 by PCR applied on several murine organ cDNAs were unsuccessful (Fig. 1, compare D and E). This observation is fitting with the exon/intron organization of the mouse gene that does not offer the equivalent of the short human exon 4 in this region of the gene (not shown). Flow cytometry revealed that CD96 is expressed by the human AML cell line KG1. Therefore, KG1 cells were surface-biotinylated and subjected to immunoprecipitations to visualize hCD96 by SDS-PAGE/Western blotting (Fig. 1F). Both the anti-hCD96 mAb TH-111 as well as recombinant hCD155-hIgG, precipitated a protein with an apparent molecular weight of ∼160 kDa. This confirms earlier observations (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar) and indicates extensive post-translational modification as already shown for murine CD96 (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). Following cloning of the full-length cDNA encoding hCD96V1 and hCD96V2, the sizes of the two variants were analyzed separately following transient expression in HEK293 cells, which are devoid of endogenous hCD96. A clear cut difference in size was not observed, even though hCD96V2 gave rise to a slightly faster migrating band. This also explains why the immunoprecipitate of biotinylated KG1 material appeared as a single band in the Western blot (Fig. 1F), although PCR indicated that both hCD96 variants are expressed by these cells (Fig. 1D). We analyzed the expression pattern of the two human CD96 isoforms in various human organs with a special emphasis on lymphoid tissue. Real time PCR revealed considerable expression of CD96 in peripheral leukocytes, thymus, lymph nodes, tonsils, spleen, as well as colon and moderate levels in ovary and kidney (Fig. 2A). An hCD96-specific message could not be detected in prostate (not shown) and brain. Among fetal tissues, hCD96 was found to be expressed in thymus and spleen at levels comparable with the adult organs and to a considerably lesser extent in kidney and brain. Because we failed to detect hCD96 in adult brain, this may indicate that hCD96 is expressed exclusively during distinct developmental stages. hCD96 was reported to be expressed on T cells, a subpopulation of B cells, and NK cells but not on monocytes and granulocytes. Therefore, white blood cells from healthy donors were isolated, sorted by flow cytometry, and/or magnetic bead separation (see “Experimental Procedures”) and the mRNA levels representing the two hCD96 variants determined in CD19+ B cells, CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD14+ monocytes, and CD3- CD56+ NK cells (Fig. 2B). In all of the subsets studied, hCD96V2 predominated, although a considerable variation of the hCD96 levels between individual blood donors was observed, especially regarding their T cell subpopulations (see error bars in Fig. 2B). Because hCD96 was identified as tumor marker expressed in T-ALL and AML (2Gramatzki M. Ludwig W.D. Burger R. Moos P. Rohwer P. Grunert C. Sendler A. Kalden J.R. Andreesen R. Henschke F. Moldenhauer G. Exp. Hematol.. 1998; 26: 1209-1214Google Scholar, 3Hosen N. Park C.Y. Tatsumi N. Oji Y. Sugiyama H. Gramatzki M. Krensky A.M. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 11008-11013Google Scholar), it was of interest to test the hCD96 expression pattern in a panel of samples representing these types of leukemias (Fig. 2, C and D). Confirming earlier results, we observed a broad range of expression levels among both AML and T-ALL samples. However, in all of the T-ALL tumors analyzed, hCD96V2 represents the major hCD96 isoform to an extent as already seen for healthy T cells. In AML cells, the general expression level of CD96 seem more moderate when compared with T-ALL, even though also the AML panel is distinguished by a huge variability regarding the individual CD96 expression levels. In contrast to T-ALL, one AML sample (Fig. 2C, A3) displayed a marked switch in the isoform expression pattern usually observed with variant 1 predominating. It has been reported that hCD96 protein levels increase several days following either allogenic or phytohemagglutinin-mediated stimulation, thereby coining the original name of CD96: Tactile (T cell activation, increased late expression) (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). To analyze whether the splicing of hCD96 pre-mRNA is influenced during T cell stimulation, peripheral T cells treated with phytohemagglutinin or anti-CD3 mAb OKT3 were studied. hCD96 protein expression was down-regulated within the first 2–3 days, but at later time points, a significant up-regulation was observed (not shown), thereby confirming earlier results (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). This particular kinetic of regulation was paralleled by the mRNA levels, although these declined earlier and to more pronounced degree. Again, the hCD96 variant specific pattern remained unchanged with hCD96V2 message predominating by a factor of ∼10 at all time points investigated (not shown). The First Ig-like Domain of CD96 Is Necessary for Binding to CD155—We next intended to determine the location of the binding epitopes on CD96 responsible for docking onto CD155 and nec-1. The finding that the CD155/CD96 interaction is not operating across the human/mouse species barrier (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar) was exploited to construct chimeric human/mouse CD96 receptors based on hCD96V2 and mCD96. To achieve this, restrictions sites were inserted into the domain 1/2 and domain 2/3 borders by site-directed mutagenesis into each receptor cDNA (Fig. 3A). Next, chimeric receptors were cloned by domain exchange (Fig. 3) and named according to their species origin (Fig. 3B). For example, receptor mhh denotes a CD96 chimera where the first human domain was replaced by its murine equivalent. In all of the constructs, the species origin of the third domain also defines that of the S/T/P-rich stalk, the transmembrane region, and the cytoplasmic tail. The receptors were then transiently expressed in HEK293 cells, and binding of recombinant mouse or human CD155-hIgG1 proteins to them was monitored (Fig. 3, D and F). In addition, the binding of various anti-CD96 mAb to the chimeric receptors was tested (Fig. 3, C and E). As expected, the anti-hCD96 mAb TH-111 did not bind at all to the murine receptor. mAb TH-111 binding to chimeric receptors suggests that epitopes residing in the first two domains are recognized. Replacement of the second human domain substantially reduced (hmm) or eliminated binding (hmh, mmh), whereas that of the first domain surprisingly caused increased antibody binding (mhh, mhm). Exchange of the third human domain had only little impact on TH-111 binding. The hCD155 binding profile to the chimeric receptors indicates that the presence of the first human domain is obligatory for the interaction with the second and possi
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