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Isolation and partial characterization of the human homologue of Thy-1.

A glycoprotein of 25,000 daltons was isolated from a human T lymphoblastoid cell line (MOLT-3). A monovalent antiserum raised against this antigen precipitated mouse Thy-1.1 antigen as well as a 25,000-dalton antigen from human blood T cells. The unabsorbed serum was also cytotoxic for both mouse and human T cells. By multiple criteria, this human T antigen appears to be homologous with mouse Thy- 1.

Island, N. Y.) plus 10% heat-inactivated horse serum. The cells were collected and disrupted as previously described (11). Nuclei and cell debris were removed by centrifugation at 400 g for 15 min. The supernate was centrifuged for 20 min at 4,000 g. The resulting supernate was centrifuged at 20,000 g for 1 h to harvest a microsomal pellet. This was resuspended in 10 mM Tris-H(21, pH 8.2, and mixed with 10 vol of acetone at -70°(2. After centrifugation at 3,000 g for 15 min, the pellet was solubilized in 2.5% deoxycholate (DO(3) as previously described (5). Nonsolubilized material was removed by centrifugation at 100,000 g for 1 h.
The solubilized antigen was then isolated by affinity chromatography followed by gel filtration. Lens culinaris lectin coupled to Sepharose 4B (Pharmacia Fine Chemicals, Div. of Pharmacia, Inc., Piscataway, N. J.) was prepared by the method of Allan et al. (12) and used to prepare a column equilibrated with 0.5% DOC, 10 mM Tris-HCl (pH 8.2) buffer. The 100,000 g supernate of the DOC-solubilized pellet was passed through this column, the unbound material was removed by washing with buffer, and the glycoproteins were then eluted with 3% alpha-methyl-D-glucoside in the same buffer. The eluted fraction was concentrated by adding 3 vol of absolute ethanol and incubating at -20°C for 48 h. The suspension was centrifuged at 300 g for 15 min, the ethanol decanted, and the pellet resuspended in a small volume of buffer. Gel filtration of the resuspended pellet was carried out on a 2.5 × 100-cm Ultrogel AcA-34 (LKB Instruments, Inc., Rockville, Md.) column in 0.5% DO(2, 10 mM Tris-HCl, pH 8.2. An antiserum was raised in rabbits against the 25,000-dalton fraction isolated from the AcA-34 column. The molecular weight of the protein peak was determined by SDS-PAGE. The first injection of antigen was administered intramuscularly in complete Freund's adjuvant. The second and third injections were given 14 and 28 d later with incomplete Freund's adjuvant. The antiserum (anti-p25) was heat inactivated and used in these experiments without any adsorptions. A goat antiserum raised against purified Thy-1 (goat anti-purified Thy-l.1 molecule [anti-p Thy-1]) isolated from a mouse AKR-derived T lymphoblastoid cell line (BW5147) was prepared as previously described (5).
Surface membrane determinants reactive with these two antisera were examined by solubi. lizing radiolabeled lymphocyte membrane antigens in 0.5% DOC and then coprecipitating them with antiserum and Staphylococcus aureus (2owan strain I (Staph protein A) as previously described (10). They were then analyzed by SDS-PAGE on a 5-12% gradient gel in a discontinuous buffer system using the method of Laemmli (13). The gels were sliced into 1-mm slices and counted on a Beckman Bio-Gamma gamma counter (Beckman Instruments, Inc., Fullerton, Calif.). Relative mobilities (Rf) were expressed as the ratio of the length of migration of the sample and a bromophenol blue dye marker. Known proteins used for estimation of molecular weight were bovine serum albumin (BSA) (68,000), ovalbumin (45,000), chymotrypsinogen A (25,000), and myogtobin (17,000). A standard curve was used to estimate the molecular weights of the antigens.

Results
Solubilized membranes of human lymphocytes were treated with unabsorbed anti-p25 serum raised against purified human T-cell antigen. Anti-p25 precipitated only one protein of 25,000 daltons from E-rosette-positive T cells from human blood ( Fig.  1 A). No labeled material was precipitated from solubilized membranes of a B-cell leukemia (pre-B-cell type). Cross-reactivity between the human and mouse T-cell antigens was demonstrated in experiments in which anti-p25 was used to precipitate purified radiolabeled mouse Thy-1.1 antigen (Fig. 1 A). Neither the prebleed normal rabbit serum and Staph protein A nor Staph protein A alone precipitated Thy-l.1 antigen. Immunodepletion experiments were then performed to determine whether the anti-p25 serum and the anti-p Thy-1 serum were recognizing the same antigenic component. When an excess of anti-p25 serum plus Staph protein A was first used to coprecipitate purified Thy-l.1, no additional material was precipitated from the depleted supernate by a second precipitation step with anti-p Thy-l.1 plus Staph protein A (Fig. 1B). Conversely, rabbit anti-p25 did not precipitate Thy-1 protein from preparations previously reacted with anti-p Thy-1 (data not shown, but identical to Fig. 1 B). The cross-species reactivity of anti-p25 for mouse T lymphocytes was also determined with a SlCr release cytotoxicity assay (5). Anti-p25 serum was cytotoxic for virtually all AKR/Jax (The Jackson Laboratory, Bar Harbor, Maine) mouse thymocytes in the presence of guinea pig complement, but was unreactive with mouse bone marrow cells (Fig. 2). The same results were obtained with goat anti-p Thy-1.1. The 50% cytotoxicity titer of anti-p25 was 1:8 for human T lymphoblastoid target cells (MOLT-3) and 1:64 for cuhured mouse T cells (BW5147) in the presence of rabbit serum as a complement source. No lysis of MOLT-3 cells occurred with guinea pig serum as a complement source. Reactivity of anti-p25 serum with MOLT-3 ceils was also demonstrated by a direct immunofluorescence assay (10).

Maximum counts released by acid. lysis -counts released in complement control
This experiment is representative of three individual studies that show that anti-p25 serum crossreacts with mouse thymocytes but is unreactive with marrow lymphoid cells. 403 membranes (not detergent solubilized) obtained by differential centrifugation (5). Before adsorptions, each antiserum was diluted to a titer that was not in antibody excess but was still cytotoxic for >50% ofAKR/Jax thymocytes as previously described (5). 50 #1 of antiserum at the appropriate dilution was incubated with an equal volume of serial dilutions of the adsorbing material for 2 h at 4°C. The membrane protein concentration of each adsorbing material was determined by the method of Lowry et al. (14) with BSA as a standard. The results confirmed again that there was cross-reactivity of the human and murine T antigens, because membranes or purified antigens from either species reciprocally removed the cytoxic activity of both antisera for AKR thymocytes (Fig. 3). There were quantitative differences in the adsorbing efficiency of the various preparations. Thus, the quantity of MOLT-3 cell membranes required for complete antibody adsorption was 10-fold greater than that of AKR/Jax brain or thymocyte membrane and 100-fold greater than the quantity of purified Thy-l.1 antigen (Fig. 3).

Discussion
We have previously demonstrated cross-reacting T lymphocyte differentiation antigens among mammalian species (15). With few exceptions, no cross-reactivity of lymphocyte antigens was demonstrated between any phylogenetic order, On the other hand, serological cross-reactivity between humans and rodents has been demonstrated for histocompatibility antigens and for immunoglobulin products of B cells (16)(17)(18).
A homology between a human T-cell antigen and murine Thy-1 is suggested by the following observations: (a) the antigens have a similar mol wt of ~25,000 as determined by SDS-PAGE; (b) they are glycoproteins that bind to L. culinaris; (c) the p25 antigen from human MOLT-3 cells elicited an antiserum that cross-reacts with mouse Thy-1 both by radioimmunoprecipitation and by complement-mediated cytotoxicity; and (d) anti-p25 and anti-p Thy-1 reacted with the same antigen as demonstrated by adsorption and immunodepletion experiments. The cytotoxicity and the quantitative adsorption experiments indicate a relatively low density of Thy-1 homologue on cultured human MOLT-3 cells compared with mouse T cells and brain. This data thus supports previous suggestions (19,20) that there is a human homologue of rodent Thy-1 analogous to the classical and physicochemical description of the purified Thy-1 molecule. Whether the molecular structure of the human T antigen is identical to the already defined rat and mouse Thy-1 molecule remains to be determined.
Human T-cell membranes are known to contain a multitude of antigenic determinants with different molecular weights (21). The ability to purify and then to immunize with immunochemically defined human T-cell antigens has the advantage of producing monospecific xeno-antisera requiring little if any adsorptions or monoclonal antibodies produced by antigen-directed hybridomas. Owen and Fanger (22) have previously prepared anti-human T-cell sera by immunizing rabbits with purified antigens taken from slices of a polyacrylamide gel. The yield of antigen with such an approach is relatively low. On the other hand, human lymphocyte membrane glycoproteins can be isolated in microgram to milligram quantities as described in this report when employing a system that includes large-scale tissue culture, affinity chromatography, and gel filtration chromatography. This yield and purity should facilitate future studies of human T antigens with respect to their molecular nature and biological properties of T lymphocytes displaying them on their membrane surface.

Summary
A glycoprotein of 25,000 daltons was isolated from a human T lymphoblastoid cell line (MOLT-3). A monovalent antiserum raised against this antigen precipitated mouse Thy-1.1 antigen as well as a 25,000-dalton antigen from human blood T cells. The unabsorbed serum was also cytotoxic for both mouse and human T cells. By multiple criteria, this human T antigen appears to be homologous with mouse Thy-1.