Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b, k) and controlled by a gene locus closely linked to theAkp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i. e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78 000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing theLy-31 andAkp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.     

A structural model for the location of the Rodgers and the Chido antigenic determinants and their correlation with the human complement component C4A/C4B isotypes

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12 000–14 000, in contrast to initial studies which showed the relative mass to be 33 000. Using polymorphic Ly-6-specific antibodies, the 33 000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2⁺, Ly-6A.2^– strains and a Ly-6A.2^– mutant cell line BW(Thy-1^–e). This clearly demonstrated that 33 000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2⁺ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12000–14000, although a minor 33 000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipilate the 12000–14000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12 000–14 000 molecules were only immunoprecipitated from Ly-6A.2⁺ cells, whereas 33 000 molecules were precipitated from both Ly-6A.2⁺ cells and Ly-6A.2^– cells. These findings suggest that the 33 000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.     

Biochemical characterization of H9/25, an allospecificity encoded by the Ly-6 region

H9/25, an allospecificity encoded by the Ly-6 region, was biochemically characterized. It was sensitive to pepsin and heat treatment, but was resistant to periodate oxidation. Its apparent molecular weight was approximately 12 000 daltons by gel filtration. The antigenic molecule was partially purified by gel filtration and antibody affinity chromatography. The partially purified antigen molecule was radioiodinated, immunoprecipitated with monoclonal antibody H9/25, and analyzed by SDS-polyacrylamide gel electrophoresis. The autoradiograph showed the molecular weight of H9/25 to be approximately 15000 daltons under reducing conditions. These results indicate that H9/25 is a protein with a single polypeptide chain of 12000–15000 daltons molecular weight, and the antigenic specificity is carried by a peptide but not a carbohydrate moiety.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Activation of murine T lymphocytes with monoclonal antibodies: detection on Lyt-2+ cells of an antigen not associated with the T cell receptor complex but involved in T cell activation

A new T cell molecule defined by the mAb 143-4-2 has been identified that is involved in T cell activation. The expression of the 143-4-2-defined epitope is linked to the previously characterized Ly-6 locus and restricted to bone marrow cells and to a subset of peripheral Lyt-2+ cells. In comparison to other anti-Ly-6.2 mAb, the 143-4-2 mAb appears to be directed at an allogeneic determinant of the Ly-6.2C molecule. The anti-Ly-6.2C antibody can promote the lysis of antigen-non-bearing target cells by alloreactive CTL clones, and in the presence of cofactors (PMA or IL 2) induces a subset of Lyt-2+ cells to proliferate, perhaps through an autocrine pathway. Although the antibody described has antigen-like effects as described for anti-TcR complex reagents, studies performed with a recently derived anti-murine T3 mAb suggest that the Ly-6.2C molecule is not associated on the cell surface with components of the TcR complex. Nevertheless, cell surface expression of the TcR complex is required for optimal triggering of T cells via the Ly-6.2C molecule. Because Ly-6.2C determinants are expressed in bone marrow and not in the thymus, the possibility is considered that expression of this molecule identifies a distinct subset of extrathymically derived T cells.     

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.     

Lyb-8.2: A new B cell antigen defined and characterized with a monoclonal antibody

A DBA/1 B10.D2-specific monoclonal antibody (CY34) is described which defines a new murine B lymphocyte differentiation antigen designated Lyb-8.2. The ontogeny, strain distribution, and cell-surface density of the antigen were studied by radioimmunoassay and by fluorescence-activated cell sorter (FACS) analysis. Lyb-8.2 appears to be expressed on pre-B cells and on all mature B lymphocytes. Lyb-8.2 molecules immunoprecipitated from surface labeled B10.D2 spleen cells migrated in polyacrylamide gels with an apparent mol. wt. of 95000–105000 daltons and were bound by lentil lectin. The expression of Lyb-8.2 is controlled by a locus on chromosome 7 that is closely linked to Gpi-1 and RP-2. Added Lyb-8.2-specific antibody did not measurably impair B lymphocyte function in several in vitro systems studied.     

Studies of the mouse Ly-6 alloantigen system

Three monoclonal antibodies were produced by fusing mouse myeloma cell line NS-1 with spleen cells from C3H/An mice hyperimmunized with B6-H-2^k spleen cells. These antibodies recognized an alloantigen displaying a similar strain distribution pattern to the Ly-6.2 and Ala-1.2 alloantigens. Analysis of C×B and B×H recombinant inbred mice revealed close linkage of genes controlling Ly-m6 and Ly-6. The monoclonal antibodies lysed 70 percent of cells in lymph nodes and 60 percent in spleen in direct cytotoxicity assays, but did not lyse significant numbers of cells of thymus and bone marrow. Separated T and B cells were reactive with the antibodies, but T cells were more sensitive to the antibody and complement than B cells. Virtually all cells in cultures of cells activated in the mixed lymphocyte reaction or by Concanavalin A were reactive with the monoclonal antibodies. Direct plaque-forming cells were completely eliminated by the monoclonal antibody and complement. By absorption tests, cells from all organs tested so far (thymus, lymph node, spleen, bone marrow, brain, kidney and liver) were shown to express the Ly-m6 determinant. Tumor cell lines with T, B or stem cell characteristics were reactive with the monoclonal antibody by direct cytotoxicity and absorption assays.     

Ly-6 region regulates expression of multiple allospecificities

The relationship between two alloantigens on mouse lymphocytes, that is Ly-6.2 and H9/25, which have previously been shown to have identical strain distribution patterns, was further investigated. Analysis of 39 (AKR × CBA) × CBA backcross progeny showed no segregation between these two antigens, indicating a close genetic linkage between them. Serological analysis showed that Ly-6.2 and H9/25 are differentially expressed on T-cell hybrid lines. Furthermore, cross-absorption of anti-Ly-6.2 serum with two cell lines revealed a heterogeneity among Ly-6 specificities. Semipurified H9/25 antigen failed to block anti-Ly-6.2 serum while anti-Ly-6.2 serum did not significantly block monoclonal antibody H9/25. These results suggest the presence of multiple allospecificities encoded for by the Ly-6 region.     

Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity

In studies with alloantisera and monoclonal antibodies (mAb) a number of antigenic determinants have been defined that are the products of the Ly-6 locus on murine chromosome 2 and that are expressed primarily on B and T lymphoid cells. It remains controversial whether these antigenic determinants are encoded by a single gene or a multigene complex. We have characterized a new rat mAb, D7, which recognizes a cell surface antigen whose expression on nonactivated peripheral lymphocytes varies from strain to strain. The phenotype of the staining profile, i.e., high or low percentage of D7-positive cells, mapped to the Ly-6 locus as assayed by strain distribution studies, RI lines, and Ly-6 congenic strains. The binding of D7 to Ly-6.1-positive strains could be inhibited by mAb directed to the Ly-6E.1 specificity, whereas D7 could inhibit the binding of mAb specific for Ly-6A.2 to cells from Ly-6.2-positive strains. Coprecipitation studies followed by Western blot analysis confirmed that D7 reacts with both Ly-6E.1- and Ly-6A.2-bearing molecules. The most likely explanation for these findings is that Ly-6A.2 and Ly-6E.1 represent allelic specificities. Further dissection of the complexity of the Ly-6 antigen system and determination of its possible functional importance in lymphocyte activation should be greatly facilitated by the availability of xenogeneic mAb that recognize framework determinants on multiple Ly-6 products.     

Stimulation of T cells via the TAP molecule, a member in a family of activating proteins encoded in the Ly-6 locus

T-cell activating protein, TAP, is a Ly-6 encoded 12,000 dalton glycoprotein involved in the activation of murine T cells. TAP is distinct from other known surface activating structures, such as the T cell receptor/T3 complex and Thy-1. This study investigates the mechanism of activation via the TAP molecule. Soluble alpha TAP monoclonal antibody (MAb) is sufficient for T cell activation. This, however, requires cross-linking because F(ab) monovalent antibody is not stimulatory unless cross-linked by a second antibody. Immediately after cross-linking, there is a rapid influx of calcium, which is comparable with concanavalin A or T cell receptor triggered responses. Subsequently, interleukin 2 (IL 2) is produced, and IL 2 receptors (IL 2R) are expressed. TAP-stimulated T cell proliferation is driven by this autocrine pathway because it is inhibited by alpha IL 2R MAb. Thus TAP-mediated activation appears to share a common final pathway with other mitogenic stimuli. A nonactivating alpha TAP MAb was observed to stimulate T cells upon additional cross-linking. Given this observation, we asked whether other Ly-6 linked proteins might share similar activating potential. We show that at least three distinct Ly-6 linked molecules are capable of stimulating T hybrid clones and/or heterogeneous T lymphocytes. Thus it appears that the Ly-6 locus encodes a family of activating cell surface molecules.     

Differential epitope expression of Ly-48 (mouse leukosialin)

Ly-48 is a major sialoglycoprotei expressed on the surface of variety of mouse hematopoietic cells that exhibits many characteristics isoforms and may function in signal transduction and cell adhension. Ly-48 is recognized by the 3E8-specific monoclonal antibody (mAb) and it has been suggested that it is the same antigen recognized by another mAb known as S7. In this report, we demonstrate definitively by transfection of a Ly-48 cDNA that S7 and two previously uncharacterized mAbs, S11 and S15, recognize the same antigen as teh 3E8-specific mAb. However, 2-D gel immunoblot analyses demonstrate the complex nature of Ly-48. Although all four mAbs react similarly with lysates from the M-45 B-cell myeloma line, 2-D immunobot analyses of the EL-4 T-cell line reveal three distinct patterns of reactivity. Further, while transfection of Ly-48 into the K562 erythroleukemic cell line conferred reactivity to all four mAbs, transfection of the Ly-48 cDNA into the nonhematopoietic cell line, Line 1, conferred reactivity only to the S11 and S15 mAbs. Thus, the Line 1 transfectants suggest the importance of posttranslational modifications in the expression of the 3E8 and S7 epitopes. Interestingly , developing fetal liver cells show the same pattern of differential Ly-48-specific mAb reactivity. The developing early fetal liver cells are reactive with S11 and S15 but are negative, to very weakly, reactive with the 3E8-and S7-specific mAbs. These results show that Ly-48 epitopes can be expressed independently on cell lines in vitro and are differentially expressed on healthy cells in vivo.     

Selection of T cell receptor expression mutants through the functionally linked Ly-6A

Ly-6A is a glycosyl-phosphatidylinositol (GPI)-anchored molecule that participates in murine T cell activation. Activation of T cell hybridomas with anti-Ly-6A monoclonal antibody (mAb) leads to production of interleukin-2 (IL-2), but also to a paradoxical growth inhibition, which was used to select for signaling mutants. Fifteen subclones derived from two independent mutageneses and anti-Ly-6A selection were characterized. Thirteen subclones responded poorly or not at all to soluble anti-Ly-6A mAb. Although the selective pressure was exerted through Ly-6A, only one mutant did not express the Ly-6A antigen. Interestingly, 10 of the 15 subclones expressed either nondetectable or a very low level of T cell receptor/CD3 complex (TCR/CD3). Preferential expansion of TCR/CD3 expression mutants following anti-Ly-6A selection further established functional linkage between Ly-6A and TCR/CD3 complex. The mechanism of the functional coupling was investigated by analyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), one of the early events in T cell activation. We showed that PIP2 was not hydrolyzed in response to anti-Ly-6A in TCR/CD3-negative mutants. Aluminum fluoride, which activates G protein directly, did induce PIP2 hydrolysis in these cells. These data suggest that activation signals originated from Ly-6A must be transmitted first to TCR/CD3 complex, which then couples to the G protein/phospholipase C system. A similar requirement also applies to the Thy-1 protein and lectin receptors. Thus, the TCR/CD3 complex plays a central role in the integration and transmission of activation signals that originated from several T cell surface molecules.     

Identification of a human Ia antigen that is different from HLA-DR and DC antigens

A monoclonal antibody, MHM4, identified a cell surface antigen present on B cells and not resting T cells. It precipitated two polypeptide chains of 34 000 and 28 000 daltons from B lymphoblastoid cells. This antibody bound to all B-cell lines tested, except those homozygous for HLA-DR7. Saturation binding assays and Scatchard plots of MHM4 binding to cells that did not carry HLA-DR7 indicated that this antibody bound less than the total surface Ia antigen. When the antibody was competed with eight other HLA-D-specific antibodies, the epitope recognized was shown to be distinct. Two-dimensional gel analysis revealed that a simple pattern of spots was precipitated, unlike the complex patterns obtained with other HLA-D-specific antibodies. The α and β spots were different from those precipitated by HLA-DR- or DC-specific antibodies. It is argued that the MHM4 antigen is the product of an HLA locus that is distinct from HLA-DR and DC. Its relationship with HLA-SI3 is discussed.     

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.     

The mouse Ly-17 locus identifies a polymorphism of the Fc receptor

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab)₂ fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2AG2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.Abbreviations used in this paper Ig immunoglobulin - FcR receptor for the Fc portion of Ig - TNP trinitrophenyl - Fab antigen-binding fragment - pA Protein A - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - PBS phosphate-buffered saline - BSA bovine serum albumin - SAMIg sheep antimouse Ig - SRBC sheep red blood cells - C complement - FITC fluorescein isothiocyanate - CNBr cyanogen bromide - EA antibody-sensitized erythrocytes     

抗人表皮生长因子受体单克隆抗体的制备、鉴定及初步应用

 用人上皮癌细胞系A 431细胞作为抗原免疫BalB/c小鼠,制备七株抗人表皮生长因子受体的单克隆抗体的杂交瘤,这些杂交瘤经三次亚克隆后仍能稳定地分泌单克隆抗体。对其中四株杂交瘤分泌的单克隆抗体进行了鉴定。免疫沉淀放射自显影结果示单克隆抗体3、101和176均可识别A 431细胞膜抗原MW为170000的蛋白质即EGF受体。单克隆抗体59可以识别低分化鼻咽癌细胞膜上EGF受体。单抗3、176和59等可抑制EGF与受体的特异结合,而101和94则不能抑制EGF与受体的结合。 用Protein-A Sepharose CL4B纯化了单抗,纯化的单抗主要为IgG_1亚类。用SDS聚丙烯酰胺凝胶电泳对纯化的单抗进行了纯度测定。     

Phenotypic changes induced by interferon in resting T cells: major enhancement of Ly-6 antigen expression

The murine Ly-6 locus controls multiple cell surface antigenic specificities with distinct cellular and tissue distributions. Although the functions of Ly-6 antigens are unknown, several of these antigens represent interesting markers of T cell differentiation and activation. In this work we used a panel of monoclonal antibodies (MAb) in conjunction with flow cytofluorometry (FCF) analysis to investigate the effect of interferon (IFN) on the surface representation of T cell-associated Ly-6 antigens. It was found that in vitro treatment of purified T cells from both C57Bl/6 (Ly-6.2) and BALB/c (Ly-6.1) mice with 10 to 10(4) U IFN-alpha/beta/ml results in a dose-dependent enhancement of Ly-6 antigen expression. This effect was already detectable after 12 to 18 hr and culminated after 48 hr of incubation. Both frequencies and brightness of Ly-6 bearing cells were increased. The most dramatic shifts were observed for the Ly-6A, D, and E antigens, which were augmented by eightfold to 20-fold upon exposure to 10(4) U IFN alpha/beta/ml. Expression of Ly-6C antigens was enhanced by fourfold to sixfold under the same conditions. Immunochemical analyses and use of metabolic inhibitors additionally demonstrated that such IFN-alpha/beta-induced phenotypic alterations of T cells reflect augmented de novo biosynthesis of Ly-6 molecules. Comparison of purified IFN-alpha and IFN-beta revealed that both are equally active in influencing Ly-6. IFN-gamma also enhanced Ly-6 expression but less efficiently than IFN-alpha/beta. Additional experiments were carried out to determine the selectivity of IFN-alpha/beta action on T cell phenotype. These studies demonstrated that IFN-induced Ly-6 enhancement occurs without emergence of interleukin 2 or transferrin receptors. Expression of H-2 and beta 2m antigens, previously known to be sensitive to IFN, was increased but to a much lesser extent than Ly-6. Most other cell surface antigens examined were minimally affected by IFN-alpha/beta with the exception of Ly-11 and Ly-23. Augmentation of these latter markers was lower than for Ly-6 antigens, however. Therefore the Ly-6 locus appears to be preferentially activated by IFN-alpha/beta in resting T cells. Additional exploration of this phenomenon should provide insight into both the biological significance of Ly-6 antigens and the mechanism(s) by which IFN affect T cell functions.