Monoclonal Antibody F11 Research Articles

Activation of resting T lymphocytes by a monoclonal antibody directed against an allotypic determinant on the T cell receptor

The murine monoclonal antibody F23.1 reacts with an allotypic determinant on the beta chain of the T cell receptor expressed by approximately 20% of T helper and cytotoxic T lymphocytes of most common mouse strains. This IgG2a antibody, either in soluble form or covalently coupled to Sepharose beads, can activate resting T cells from naive animals to proliferate. Interestingly, under all conditions of activation, the antibody can only induce proliferation if exogenous lymphokines in the form of Con A supernatant are provided. Thus, it is unlike most lectins and anti-T3 antibodies in this regard. Furthermore, under all conditions of culture, F23.1 activates preferentially the Lyt-2+ subset of T cells. This is the case even in the presence of accessory cells. Further evidence is provided that two soluble lymphokines, different from IL2, are required to initiate IL2-dependent growth and to allow the expression of lytic activity.

The in vitro regulation of human thyrocyte HLA-DR antigen expression.

The ability of endocrine organs to express human immune response-associated antigens (Ia), such as HLA-DR, is a subject of intense current interest. In this study, the effects of various potential modulators of thyroid follicular cell HLA-DR expression were examined using in vitro cultures. A culture supernatant containing T-cell-derived lymphokines caused DR antigen expression on 13-18% of thyroid cells; more consistent effects were produced by recombinant gamma-interferon, which led to 46-100% of the thyroid cells becoming HLA-DR positive after 3 days in culture. This effect was both time and concentration dependent and occurred in thyroid cells derived from patients with Graves' disease (n = 7) and Hashimoto's thyroiditis (n = 2) as well as from three subjects with no autoimmune thyroid disease. Thyroid cells stained with the monoclonal antibodies 4F2 and 5E9, which recognize cell activation antigens, regardless of whether they were treated with gamma-interferon. The lectin phytohemagglutinin also induced HLA-DR antigen expression (21-91% of cells positive). This response was dependent on T cell contamination of thyroid cell suspensions, since the effect was inhibited by cyclosporin A. HLA-DQ antigen expression, identified by the Leu-10 monoclonal antibody, was also induced on thyroid cells by gamma-interferon and phytohemagglutinin. In contrast, neither recombinant alpha-interferon nor interleukin-2 induced HLA-DR antigens. Irradiation reduced the response of thyroid cells to gamma-interferon, but two of the known inhibitors of macrophage Ia expression, prostaglandin E2 and (Bu)2cAMP, did not affect gamma-interferon-induced thyroid cell HLA-DR expression. We were unable to detect interleukin-1 production by thyroid cells. These results suggest that 1) under normal circumstances, thyroid cells are 4F2 and 5E9 positive, but are incapable of expressing Ia antigens and, thus, of activating T cells to initiate autoimmune thyroiditis; and 2) once activated, for example by a virus, T cells could release gamma-interferon and induce thyroid cell HLA-DR and -DQ antigen expression; these Ia-positive thyroid cells could then have a role in maintaining or enhancing the autoimmune response.

Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor.

Repeated immunization of normal C57L/J (H-2b) mice with peripheral T cells from BALB.B (H-2b) mice results in the production of antibodies which react with the T cell receptor. A monoclonal antibody-producing hybridoma, F23.1, was isolated from immunized C57L/J mice showing this property. This monoclonal antibody recognizes approximately 25% of peripheral T cells in BALB mice. It stains approximately the same fraction of T cells and precipitates the same heterodimer as the rat monoclonal antibody described previously that was made against isolated receptor material. The allotypic determinant recognized by this monoclonal antibody is present in most common laboratory strains (BALB, C57BL, CBA, A, DBA, C3H) and is absent in C57L, C57BR, and SJL mice. Sorting peripheral T cells from BALB.B or (SJL X BALB/c)F1 mice for the F23.1+ and F23.1- subsets revealed that both populations contain approximately the same CTL precursor frequency for alloantigen. Thus, the T cell receptor allotype defined by F23.1 is present on CTL. Furthermore, cytotoxicity mediated by an F23.1+ CTL line could be blocked specifically by the F23.1 monoclonal antibody. Under appropriate conditions, the monoclonal antibody F23.1 bound to Sepharose 4B beads can induce resting peripheral T lymphocytes of allotype-positive strains to proliferate.

Cytotoxic T lymphocyte-mediated lysis via the Fc receptor of target cells.

The murine monoclonal antibody F23.1 reacts with an allotypic determinant on the T cell receptors of about 25% of peripheral T lymphocytes in most common mouse strains. In this report we demonstrate that this IgG2a antibody bound to the receptor of a cytotoxic effector T cell can direct that cell to lyse any target cell which expresses receptors for IgG2a. Thus, allotype-positive, Lyt-2+ T lymphocytes from either concanavalin A- or F23.1-activated cell cultures are recruited as cytotoxic effector cells which lyse targets via the Fc receptor. Concanavalin A-induced T cell blasts depleted of Lyt-2+ cells do not mediate cytotoxic effects in this system. The data presented here confirm earlier observations that monoclonal anti-T cell receptor antibodies attached to the surface of a cell can act as target structures for T cell-mediated lysis.

Monoclonal Antibody 4F2 Reactive with Basal Layer Keratinocytes: Studies in the Normal and a Hyperproliferative State

To establish a method for separating different keratinocyte subpopulations in the epidermis, we studied the specificity of monoclonal antibody 4F2 for keratinocytes. Preliminary screening experiments had previously demonstrated 4F2 reactivity with the epidermis. 4F2 reacted with a subpopulation (19.29 +/- 5.23%) of human epidermal cells in suspension. The membrane antigen identified by 4F2 continues to be expressed by cultured keratinocytes. In frozen tissue section using an indirect immunofluorescence technique, the 4F2-positive cells in the basal layer are sharply demarcated from the negative suprabasilar layers. Even in the hyperproliferative state of psoriasis, the 4F2 reactivity is confined to the basal layer. Cell suspensions of psoriatic epidermis demonstrated a greater percentage of reactivity with 4F2 (49.51% +/- 6.50%), probably reflecting the expanded population of basal layer cells. Monoclonal 4F2, therefore, reacts with a membrane antigen present on basal keratinocytes, and provides a probe for use in the isolation of the basal keratinocyte subpopulation. Thus, this antibody should be useful in studies of normal and aberrant differentiation of the epidermis.

Ia+ T cells in new onset Graves' disease.

The Ia (immune-associated, DR) antigen is a cell surface glycoprotein which is absent on normal circulating T lymphocytes but present on activated T lymphocytes. We studied the expression of this antigen on circulating T lymphocytes from patients with untreated hyperthyroid Graves' disease. All patients (n = 33) with recent onset hyperthyroid Graves' disease studied had an increased percentage and number of circulating Ia+ T cells. Patients with non-Graves's hyperthyroidism or Graves' disease patients more than 1 yr after thyroid ablation had normal values for Ia+ T cells. Other cell surface activation antigens, recognized by monoclonal antibodies 4F2 and 5E9, were not present on circulating T cells in patients with Graves' disease. The 100% positivity for increased numbers of Ia antigen-bearing T cells in hyperthyroid Graves' disease contrasts with our finding of 70% positivity in another HLA-DR-associated disease, recent onset type I diabetes mellitus. The pathogenic significance of these cells is not known, but they seem to represent selective activation of the immune system in patients with untreated hyperthyroid Graves' disease.

Differential expression of cell activation markers after stimulation of resting human B lymphocytes.

The interaction of antigen with surface Ig receptors initiates a complex process by which resting B lymphocytes are activated. In this study, small resting B lymphocytes were isolated by countercurrent elutriation of tonsillar B lymphocytes and stimulated with a range of concentrations of anti-mu and B cell growth factor (BCGF). The subsequent expression of two glycoproteins known to be absent from resting B lymphocytes but present on activated cells were analyzed with the monoclonal antibodies 4F2 and 5E9. The antigen recognized by the 4F2 monoclonal was expressed early in the G1 phase of the cell cycle and correlated with blast transformation; this was demonstrated by stimulating resting B cells with a low concentration of anti-mu sufficient for cellular activation but not proliferation. The 5E9-defined antigen was not present unless the B cells were stimulated with a higher concentration of anti-mu or a BCGF was added to the low concentration of anti-mu. The addition of hydroxyurea to culture blocked the entrance of stimulated B cells into S phase but did not interfere with the expression of either 4F2 or 5E9. Thus, whereas the monoclonal antibody 4F2 recognized cells in early G1, 5E9 recognized cells in late G1. By double immunofluorescence staining with propidium iodide and either 4F2 or 5E9, all cells in S, G2, and M phases were demonstrated to be both 4F2- and 5E9-positive. The monoclonal antibodies 4F2 and 5E9 can be used to distinguish an activated from a resting human B lymphocyte and to delineate sequential steps in the activation process.

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation.

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11.

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.

Genetic and biochemical characterization of a human surface determinant on somatic cell hybrids: The 4F2 antigen

We have mapped the gene which codes the species-specific determinant defined by monoclonal antibody 4F2 to human chromosome 11. All human chromosomes, except Y, were included in a group of four human-mouse hybrid lines. Hybrids heterogeneous for 4F2 antigen expression were sorted using the fluorescence-activated cell sorter (FACS) to yield populations homogeneous with respect to the presence or absence of this determinant. Isozyme analysis indicated corresponding genetic selection for or against human chromosome 11. This map assignment was confirmed using a hybrid line which contained only human chromosome 11. Immunoprecipitation of the 4F2 determinant from the 11 only hybrid resulted in a heavy subunit of molecular weight (Mr) = 100,000 and a light subunit of Mr = 41,000. This contrasts with results obtained from nonhybrid human cells of different lineages. These results demonstrate the importance of FACS techniques in the rapid mapping of genes which code human cell surface antigens.

Characterization of antigen recognized by the monoclonal antibody (4F2): different molecular forms on human T and B lymphoblastoid cell lines.

The monoclonal antibody 4F2 recognizes a disulfide-linked ricin-binding glycoprotein complex (Mr congruent to 125,000) composed of a sialylated heavy subunit (Mr congruent to 85,000 on T cell lines) and an unsialylated light subunit (Mr congruent to 41,000). The antigen (T85,41) recognized by 4F2 on T cell lines is structurally distinct from the antigen (B93, 41) on B cell lines. The heavy subunits, but not the light subunits, from all T cell lines examined were uniformly smaller in size than the heavy subunits from several B cell lines. This reflects differences in carbohydrate rather than protein represent in B93,41 compared with T85,41, because both heavy subunits have a common unglycosylated form (p65) and a common partially glycosylated precursor form (p68). Among non-T, non-B hematopoietic cell lines, the monocytoid line U-937 expressed an antigen that resembles B93,41, whereas the erythroleukemic line K-562 expressed an antigen more similar to T85,41. 4F2 recognizes a protein determinant on the heavy subunit (with or without N-linked glycosylation) and also the unglycosylated heavy subunit retains the ability to associate with light subunit. The light subunit itself contains no detectable N-linked carbohydrate. Unlike the transferrin receptor, synthesis of the antigen recognized by 4F2 on the promyelocytic cell line HL-60 did not diminish upon dimethylsulfoxide-induced differentiation, and thus is not tightly correlated with cell proliferation.

Immunochemical and biosynthetic analysis of monoclonal antibody-defined melanoma-associated antigen.

Immunoprecipitation studies with application of monoclonal antibody F11 originally made to a partially purified spent medium antigen of melanoma cells, made it possible to delineate the molecular profiles of both the cell associated and spent medium antigens recognized on melanoma cells intrinsically labeled with glycoprotein precursors. F11 distinguishes a glycoprotein of Mr 100,000 (100 K) in the spent media of melanoma cells while a parallel analysis of detergent lysates of cells reveals a pattern of three glycoproteins of Mr 75, 77, and 100 K. Pulse-chase analysis of the biosynthesis of these antigens indicated that F11 first recognizes the 75 and 77 K antigens in the absence of a 100K component suggesting strongly that these molecules contain an antigenic site recognized by F11. The 100 K antigen appears later in the pulse-chase analysis with kinetics that suggest some of the 75 and 77 K antigens are biosynthetic precursors of the 100 K antigen. This molecule is ultimately secreted into the extracellular media and appears to be a sialoglycoprotein judging from its sensitivity to neuraminidase. A cross-reactive species with an approximate Mr 90 K is also recognized by F11 in indirect immunoprecipitation analysis of spent media from a neuroblastoma cell line indicating that a common antigenic site exists on this secreted but structurally different neuroblastoma antigen. Thus, a combination of immunochemical and biosynthetic analyses of cell-associated and secreted antigens recognized by monoclonal antibody F11 demonstrate such molecules can differ structurally when isolated from the same or different tumor cells. These findings indicate the necessity to establish molecular profiles of melanoma-associated glycoprotein antigens recognized by monoclonal antibodies to characterize and define their potential biological functions within tumor cells.

Human T cell proteins recognized by rabbit heteroantisera and monoclonal antibodies

Many structural and functional features of the cell surface antigens of human T cells remain to be characterized. In these investigations, heteroantisera have been used to identify potentially interesting antigens not yet recognized by individual monoclonal antibody preparations. These antigens include patterns of bands ( M r 140−240K) on various T cells and T cell lines, at least six other bands on T cell lines and novel antigens on CTL lines. The majority of these antigens are different from the compiled list of antigens recognized by monoclonal antibodies against T cell proteins. The monoclonal OKT9 recognizes an antigen most prevalent on dividing cells, and this antigen is the same as that recognized by another monoclonal antibody, 5E9, and by previously described heteroantisera. The monoclonal antibody 4F2 has a distribution similar to OKT9, but it is structurally distinct, and is also present on monocytes. Three T cell surface proteins, two defined by the monoclonal antibodies OKT6 and OKT10, recognize proteins which resemble HLA antigens physiochemically. OKT6 and NAI/34 both recognize a 49,000 glycoprotein which is associated with a 12,000 subunit which reacts with anti- β 2-microglobulin antibodies. OKT10 recognizes a 46,000 protein, also found with a 12,000 peptide, while the third protein, a β 2-microglobulin-associated glycoprotein of 43,000 is not yet defined by a monoclonal Ab. Other monoclonals described here include 3A1 and PVR-11, which are on subsets of peripheral T cell, and B 1-192 and B 1-499 which are on some activated T cells.