Three cell surface antigens associated with the cytolytic T lymphocyte(CTL)-target cell interaction were identified by generation of monoclonal antibodies (MAb) against OKT4+, HLA-DR-specific CTL and selection for inhibition of cytolysis in a 51Cr-release assay. These MAb block cytolysis by both OKT4+ and OKT8+ CTL and the proliferative responses to PHA and the mixed lymphocyte response (MLR). LFA-1 is an antigen widely distributed on lymphoid tissues and is composed of two polypeptides of 177,000 and 95,000 Mr on all cell types studied. Anti-LFA-1 MAb block NK cell-mediated cytolysis in addition to T lymphocyte-mediated cytotoxicity and proliferation. LFA-2 (Mr = 55,000 to 47,000), a determinant on the sheep red blood cell receptor, is expressed by T cells but not B cells and appears specific for T cell functions. LFA-3 (Mr = 60,000) is a widely distributed antigen present on both hematopoietic and nonhematopoietic tissues and appears to only be involved in T cell functions. MAb to LFA-1 and LFA-2 inhibit function by binding to effector cell surface molecules, whereas anti-LFA-3 MAb appear to block by binding to the target cells. Together with previously described molecules, LFA-1, LFA-2, and LFA-3 demonstrate the complexity of CTL-mediated cytotoxicity at the molecular level.
The human lymphocyte function-associated antigen-1 (LFA-1), the complement receptor-associated OKM1 molecule, and a previously undescribed molecule termed p150,95, have been found to be structurally and antigenically related. Each antigen contains an alpha- and beta-subunit noncovalently associated in an alpha 1 beta 1-structure as shown by cross-linking experiments. LFA-1, OKM1, and p150,95 alpha-subunit designations and their molecular weights are alpha L = 177,000 Mr, alpha M = 165,000 Mr, and alpha X = 150,000 Mr, respectively. The beta-subunits are all = 95,000 Mr. Some MAb precipitated only LFA-1, others only OKM1, and another precipitates all three antigens. The specificity of these MAb for particular subunits was examined after subunit dissociation by high pH. MAb specific for LFA-1 or OKM1 bind to the alpha L- or alpha M-subunits, respectively, while the cross-reactive MAb binds to the beta-subunits. Coprecipitation experiments with intact alpha 1 beta 1-complexes showed anti-alpha and anti-beta MAb can precipitate the same molecules. In two-dimensional (2D) isoelectric focusing-SDS-PAGE, the alpha subunits of the three antigens are distinct, while the beta-subunits are identical. Biosynthesis experiments showed alpha L, alpha M, and alpha X are synthesized from distinct precursors, as is beta. The three antigens differ in expression on lymphocytes, granulocytes, and monocytes. During maturation of the monoblast-like U937 line, alpha M and alpha X are upregulated and alpha L is downregulated. Some MAb to the alpha subunit of OKM1 inhibited the complement receptor type three. LFA-1, OKM1, and p150,95 constitute a novel family of functionally important human leukocyte antigens that share a common beta-subunit.
Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.
The human monoblast leukemia line, U937, is growth-inhibited and induced to develop markers of mature monocytes by lymphokine preparations. Lymphokine is cytostatic and induces expression of Fc receptors in U937 and in myelomonocytic leukemic lines RC-2A and KG-1, but does not have these effects on T- and B-lymphocytic lines. In addition to previously described properties, including complement receptors, phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC), Mac-1 and Mac-3 surface antigens defined by monoclonal antibodies are induced on U937 cells by lymphokine and phorbol ester. The Mac-1 surface component appears to have a regulatory role in differentiation of the monocyte lineage line, since antibodies to this antigen block the induction of Mac-3 antigen. The lymphokine activity was concentrated by salt precipitation and characterized by ion-exchange and size chromatography. Fractions of about 40,000 daltons were responsible for growth inhibition and induction of Fc receptors and Mac-1 antigen in U937 cells. However, ADCC was not induced in U937 by individual fractions of lymphokine, suggesting that this cytotoxic capacity may be regulated by a lymphokine of a different size, which is only effective after initial maturation steps. Since gamma-interferon is present on the 40K size range of lymphokine, the possibility that interferon is a differentiation modulator for the monoblast cells was investigated. Highly purified gamma-interferon (10(7) U/mg protein) at 10-300 U/ml inhibited growth and induced Fc receptors in U937 similar to the effect of lymphokine. The Fc-receptor-inducing activity of lymphokine was inhibited by a neutralizing monoclonal antibody to gamma-interferon, suggesting that this differentiation factor in lymphokine is gamma-interferon.
Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.
To define the characteristics and target antigens (Ags) of nephrotoxic antibodies (Abs) and to analyze the factors that govern the evolution of Ab-mediated glomerular injury, we have prepared monoclonal Abs against rat glomerular Ags. BALB/c mice were immunized with Lewis rat cortex or glomeruli, and their spleens were removed and fused with hypoxanthine-aminopterin-thymidine supplement-sensitive myelomas. Hybrids were selected for production of Abs against Lewis rat kidney by indirect immunofluorescence. To date, more than 50 positive hybrids have been selected and their tissue reactivity defined by indirect immunofluorescence and immunoelectron microscopy. Of these, 14 are presented here in detail. One of these monoclonal Abs, K9/4, recognizes a unique Ag present exclusively on the cell surface of rat glomerular visceral epithelial cells. Four Abs (K12/2, K17/4, K12/5, and K12/8) recognize sites within the glomerular basement membrane; K12/2 and K17/4 also bind to vascular basement membranes of the rat, whereas K12/5 and K12/8 bind to glomerular basement membrane, tubular basement membranes, and vascular and epithelial basement membranes in all tissues of the rat. Two hybridomas (K6/1 and K6/3) recognize determinant(s) present on cell surfaces of endothelial and epithelial cells as well as within the glomerular basement membrane. All of these previously mentioned Abs are species restricted (i.e., they bind only to rat tissue) and, with the exception of K9/4, bind upon in vivo administration. Several others, however, recognize ubiquitous Ags that are present on intracellular structures in every species tested. The tissue distribution of these Ags suggests that they are present in contractile or cytoskeletal elements and, as expected from their intracellular location, monoclonal Abs directed against these components do not bind upon in vivo administration. Future studies will be directed at defining the antigenic composition of the glomerular capillary wall and the relevance of such Ags in immune-mediated glomerular injury.
Specific antibody-secreting hybridomas have been obtained by fusing Syrian or Armenian hamster (Mesocricetus auratus or Cricetulus migratorius) spleen cells with mouse myeloma cells. The hamsters were immunized to mouse cytolytic T lymphocytes. Hybrids were selected either by an indirect binding assay using an 125I-monoclonal antibody (MAb) reactive with hamster kappa-chains or by their ability to block T cell-mediated cytolysis. Three hybridoma clones were obtained that secreted intact IgM-like and IgG-like hamster MAb as shown by SDS-PAGE. The clones were stable as shown by subcloning. Two MAb recognized antigens of wide tissue distribution; the third bound specifically to T lymphocytes, gave strong inhibition of T cell-mediated cytolysis, and immunoprecipitated the Lyt-2,3 molecule.
Langerhans cells are Ia-bearing antigen-presenting cells in the epidermis that share many functions with macrophages. We have used monoclonal antibodies to the macrophage antigens, Mac-2 and-3, Ia antigen, Fc fragment receptor and the common leukocyte antigen CLA to compare the cell surface antigens of these cells with those of interdigitating and follicular dendritic cells and of macrophages in lymphoid tissues. Immunoperoxidase staining was carried out with epidermal sheets from BALB/c mice and epidermal cell suspensions enriched for Langerhans cells by Fc rosetting. Langerhans cells stained for all of these antigens. Comparison with the staining properties of other dendritic cells and macrophages, in combination with previous observations, indicates a close relationship of Langerhans cells to the interdigitating cells of lymphoid tissues.
Mac-3 is a mouse macrophage differentiation antigen defined by a rat anti-mouse monoclonal antibody (MAb),M3/84. The structure, biosynthesis, quantitative surface expression, and distribution of Mac-3 have been studied by radiolabeling and isolation with MAb-Sepharose, saturation binding, absorption, and immunofluorescence flow cytometry. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mac-3 migrates as a diffuse band with average Mr = 110,000. Labeling of intact cells with 125I and accessibility to MAb show it is present at least in part on the cell surface. Saturation labeling with 125I-MAb shows 4.2 X 10(4) cell surface sites on thioglycollate medium-elicited peritoneal macrophages. [35S]Methionine and [3H]glucosamine incorporation into Mac-3 by purified macrophages show it is a glycoprotein synthesized by these cells. Absorption shows Mac-3 is strongest in macrophages, present in lower quantities in lung, liver, bone marrow, and spleen, and undetectable in thymus, lymph node, brain, and heart. Immunofluorescent flow cytometry shows surface expression on thioglycollate-elicited macrophages but not bone marrow, spleen, lymph node, or thymus cell suspensions. Similar amounts of Mac-3 are immunoprecipitated from resident macrophages or macrophages elicited by sterile inflammatory agents, intracellular parasites, or immunomodulators, but the average Mr of Mac-3 varies from 92,000 to 110,000. Mac-3 is synthesized from precursor(s) of Mr = 74,000 and 79,000, identical in the different macrophages. Processing into the mature molecule, which migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a more diffuse band and varies in Mr among macrophage elicited by different agents and to a lesser degree between different preparations of the same type of macrophage, occurs in 15 to 30 min.
Anti-Mac-1 (M1/70), a rat monoclonal antibody that reacts with mouse and human macrophages, polymorphonuclear leukocytes (PMNL), and natural killer cells, selectively inhibited complement receptor-mediated rosetting by murine macrophages and human PMNL. Preincubation of macrophages with anti-Mac-1 inhibited formation of rosettes with sheep erythrocytes bearing IgM antibody and murine C3 fragments. No inhibition was observed when other monoclonal antibodies that react with macrophages (such as anti-Ly5, anti-H-2, or anti-pan-leukocyte) were tested at 10-fold higher concentrations. Anti-Mac-1 did not affect macrophage Fc receptor-mediated rosetting. Erythrocytes bearing homogeneous human C3 fragments C3b (EC3b) or C3bi (EC3bi) were used to test the specificity of the murine macrophage and human PMNL complement receptor inhibited by anti-Mac-1. In both cases, anti-Mac-1 inhibited CR3-mediated rosetting of EC3bi but not CR1-dependent rosetting of EC3b. The results show that Mac-1 is either identical to CR3 or closely associated with CR3 function. This is one of the first cases in which a monoclonal antibody-defined differentiation antigen has been associated with a specific cell surface function.
Two monoclonal antibodies, M3/31 and M3/38, were obtained by fusion of mouse myeloma cells with rat spleen cells immunized to immunoadsorbent-purified macrophage glycoproteins. Co-precipitation experiments show that antigenic determinants recognized by these two antibodies reside on the same molecular species, termed Mac-2, Mac-2, an antigen of 32,000 Mr, is synthesized by and expressed on the surface of thioglycollate-elicited macrophages as shown by [35S]-methionine and 125I labeling. Saturation binding experiments show that thioglycollate-elicited macrophages express 1.7 X 10(5) Mac-2 sites/cell. Thioglycollate-elicited macrophages are strongly absorptive for 125I-labeled M3/38 MAb. Kidneys are also absorptive; however, evidence is presented pointing to the nonspecificity of this absorption. Lymph node and thymus are negative, whereas spleen and bone marrow are weakly absorptive, probably due to stromal cells. Nonlymphoid tissues, such as lung, liver, heart, and brain, exhibit slight or no absorbing capacity. Cell suspensions from spleen, bone marrow, thymus, and peripheral lymph node are greater than 99% Mac-2- by immunofluorescent flow cytometry. In contrast, thioglycollate-elicited macrophages are greater than 96% strongly positive for Mac-2. Only 20% of peptone-elicited cells are weakly positive, whereas resident peritoneal macrophages and other macrophage elicited by Listeria monocytogenes, Con A, or LPS are greater than 98% negative. SDS-PAGE of [35S]-methionine-labeled Mac-2 shows that thioglycollate-elicited macrophages synthesize 10- to 30-fold more Mac-2 than other peritoneal macrophage subpopulations, whereas all types of peritoneal macrophages synthesize and express on their surfaces similar amounts of the Mac-1 antigen. Mac-2 antigen is therefore induced in macrophages only in response to specific differentiative signals.
During the past decade the mechanism of CTL-mediated killing has been resolved into 3 steps, and its cation requirements, and general nature have been well defined. However, biochemical understanding of the CTL-target interaction has made little progress. Recently, we have developed a monoclonal antibody (MAb) which blocks killing by binding to a previously undescribed molecule on the CTL membrane, a molecule which we therefore have termed lymphocyte function-associated antigen one (LFA-1). LFA-1 and Lyt-2,3 are the only presently identified sites for such blocking; antibodies to over a dozen other molecules expressed on the CTL do not block killing. Present evidence suggests that LFA-1 is crucial in the adhesive interaction of T cells with other cells (e.g., targets, macrophages, perhaps B cells) The continuing search for blocking MAbs provides a systematic way to link specific molecules with CTL function.
Mouse monoclonal antibodies to rat IgG were obtained by fusion of immune SJL mouse spleen cells to NSI myeloma cells. Seven monoclonal antibodies have been labeled with 125I and studied as to specificity and avidity by using a panel of rat monoclonal antibodies both as inhibitors and target antigens in soft well plate and indirect cell binding assays. All MAb were selected for high avidity of 4 X 10(7) to greater than or equal to 2 X 10(9) M-1. Four MAb were subclass-specific. RG11/39, RG7/1, and RG7/11 were absolutely specific for the Fc' region of IgG1, IgG2a, and IgG2b, respectively. RG9/6 showed specificity for the Fab' region of IgG2a but crossreacted with lower avidity with IgG2c. Three MAb reacted with rat kappa chains. RG7/9 defined a monotypic (common) kappa chain determinant. RG11/15 and RG7/7 were specific for allelic kappa 1a and kappa 1b determinants, respectively. The monotypic and kappa 1a allotypic determinants are topographically separated. The antibodies can be used as screening reagents in indirect cell binding assays. They have sensitivity similar to affinity-purified rabbit anti-rat IgG and more defined specificity. They do not crossreact with mouse or human IgG, making them particularly suitable companion reagents for rat anti-mouse or anti-human MAb. One Mab, RG7/7, strongly crossreacts with Syrian hamster IgG.