Thymocyte LFA-1 and thymic epithelial cell ICAM-1 molecules mediate binding of activated human thymocytes to thymic epithelial cells.

We have investigated the binding in vitro of activated thymocytes to thymic epithelial (TE) cells, and studied the effect of up-regulation of TE cell surface intracellular adhesion molecule 1 (ICAM-1) and HLA-DR by IFN-gamma on the ability of TE cells to bind to both resting and activated human thymocytes. TE cell binding to activated and resting thymocytes was studied by using our previously described suspension assay of TE-thymocyte conjugate formation. We found that activated mature and immature thymocytes bound maximally at 37 degrees C to IFN-gamma-treated ICAM-1+ and HLA-DR+ TE cells and this TE-activated thymocyte binding was inhibited by antibodies to LFA-1 alpha-chain (CD11a) (68.1 +/- 5.6% inhibition, p less than 0.01) and ICAM-1 (73.9 +/- 7.7% inhibition, p less than 0.05). Neither anti-HLA-DR antibody L243 nor anti-MHC class I antibody 3F10 inhibited IFN-gamma-treated TE binding to activated thymocytes. As with antibodies to LFA-3 and CD2, antibodies to LFA-1 and ICAM-1 also inhibited PHA-induced mature thymocyte activation when accessory signals were provided by TE cells in vitro. Finally, LFA-1 and ICAM-1 were expressed early on in human thymic fetal ontogeny in patterns similar to those seen in postnatal thymus. Taken together, these data suggest that resting mature and immature thymocytes bind to TE cells via the CD2/LFA-3 ligand pair, whereas activated thymocytes bind via both CD2/LFA-3 and LFA-1/ICAM-1 ligand systems. We postulate that IFN-gamma produced intrathymically may regulate TE expression of ICAM-1 and therefore potentially may regulate TE cell binding to activated thymocytes beginning in the earliest stages of human thymic development.     

Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells.

We have shown that human thymic epithelial (TE) cells produce IL-1 alpha, IL-1 beta, and TE cells bind to thymocytes by CD2 and LFA-1 molecules on thymocytes and LFA-3, ICAM-1 on TE cells. We investigated whether ligand binding to LFA-3 on human TE cells can modulate TE cell IL-1 production. First, we investigated the ability of human thymocytes to regulate IL-1 release by TE cells. Both autologous and allogenic emetine-treated thymocytes when cultured with TE cells augmented IL-1 release by TE cells. The augmentation of IL-1 release was cell density dependent. Inasmuch as the interaction between thymocytes and TE cells is mediated in part by CD2 molecules on thymocytes and LFA-3 molecules on TE cells we next determined the effect on IL-1 release of ligand binding (anti-LFA-3 mAb TS2/9) to TE cell surface LFA-3. Purified anti-LFA-3 mAb augmented IL-1 release in a concentration-dependent fashion. The anti-LFA-3-mediated augmentation of IL-1 release required both new protein and RNA synthesis as shown by the ability of cycloheximide and actinomycin-D to inhibit augmentation of IL-1 production by TE cells, and by direct quantitation of IL-1 alpha and IL-1 beta mRNA by Northern blot analysis. Both F(ab)'2 and Fab' fragments of anti-LFA-3 mAb augmented IL-1 alpha and IL-1 beta mRNA production, indicating that monovalent binding to cell surface LFA-3 was sufficient to provide the inducing signal. The identification of LFA-3, the cell surface ligand for thymocyte CD2 molecules, as a molecule via which TE cell-derived cytokine production may be regulated suggests a mechanism at the cell surface by which direct TE cell-thymocyte interaction might result in the triggering of local IL-1 release within the human thymic microenvironment.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Production of anti-thymulin (FTS) monoclonal antibodies by immunization against human thymic epithelial cells

A monoclonal antibody specific for thymulin (FTS), a thymic hormone initially isolated from serum, was obtained by cell fusion using spleen cells from BALB/c mice immunized with cultured human thymic epithelial cells. Hybridomas were selected according to their capacity to produce antibodies binding specifically to thymic epithelial cells in culture (as assessed by indirect immunofluorescence) and their ability to absorb in vitro the biological activity of synthetic and natural hormone preparations and to induce in vivo the disappearance of endogenous circulating thymulin. In this way monoclonal antibodies were obtained that recognized a subpopulation of nonlymphoid cells on frozen sections of mouse and human thymuses. The epithelial nature of these cells was assessed using an antikeratin antiserum. The binding of the antibodies to thymic cells was completely abolished by its absorption with the synthetic hormone or normal (but not of thymectomized) mouse serum. The thymic specificity of the antibody was further confirmed by the complete absence of binding to sections of all the various lymphoid and epithelial organs examined (from both humans and mice). Double labeling experiments using the monoclonal antibody described above and a monoclonal antibody prepared by immunization with the synthetic peptide showed that the two antibodies bound to the same cell. These results provide further evidence for the exclusive presence of the thymic hormone thymulin in thymic epithelial cells.     

Antigenic subsets of human breast epithelial cells distinguished by monoclonal antibodies

Three monoclonal antibodies raised to the human milk fat globule membrane bind, within the normal breast, to the surface of the luminal epithelial cells but not to the surrounding myoepithelial, connective tissue, or blood vessel cells. These antibodies distinguish three subsets of the epithelial cells that are not distinguishable by conventional histology. To show the arrangement of the cells in two dimensions over the sheet of epithelium, ducts were dissected out of normal breast tissue, opened up and laid flat as sheets of epithelium. The apical faces of the cells were strained, unfixed, using two-color immunofluorescence to contrast the subsets of cells stained by the different antibodies. The epithelium was then seen to be a mosaic of cells that express different surface antigens. The grouping and appearance of the cells stained by the different antibodies was characteristic. This may be just a random heterogeneity of antigen expression but alternatively the different cells may be in different physiological states. Regardless of its biological significance, the observation has practical consequences for the use of such antibodies in identifying cells and the study of antigenic heterogeneity in tumors.     

Response to Mycobacterium tuberculosis antigens is enhanced by monoclonal antibodies.

T lymphocytes purified from peripheral blood of tuberculosis patients and from healthy skin test PPD-positive subjects show a remarkably increased proliferative response in culture to purified protein derivative (PPD) when three anti-mycobacterial monoclonal antibodies are added together with the antigen. This indicates an important role played by antibodies in the T cell response to the tuberculous infection.     

Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells

We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.     

Glycosphingolipid carriers of carbohydrate antigens of human myeloid cells recognized by monoclonal antibodies

Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycosphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Gal beta 1----4(Fuc alpha 1----3)GlcNAc and Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-N-acetyllactosamine type that are susceptible to degradation with endo-beta-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.     

Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.     

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

Molecular properties of ECMA 2 and ECMA 3 antigens defined by monoclonal antibodies against embryonal carcinoma cells

ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other alpha-galactosyl antigens, namely blood group B and P1 antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Anti-(human LFA-1) monoclonal antibodies bind P815 murine tumour cells

Summary Using anti-CD11a and anti-CD18 monoclonal antibodies (mAbs) directed respectively against the and the chains of LFA-1, we obtained an important and specific staining of P815 murine tumour cells. Both ascitic and cultured cells displayed a positive staining. Other murine tumours of haematopoietic origin, as well as lymphocytes or lymphoblasts from DBA/2 mice, were not labelled by the same monoclonal antibodies. These results were surprising since, to our knowledge, no case of cross-reaction between species has been reported with LFA-1. Moreover, competition assays showed that epitopes recognized by the two anti-CD11a antibodies were different from those identified by H35.89.9, a mAb raised against the murine LFA-1 chain. Using allogeneic cytotoxic T lymphocytes, we also showed that anti-(human LFA-1) mAbs were unable to block the lysis of P815 by these effector cells. Thus, the putative functional properties of these structures, as well as their importance from an antigeneic point of view, remain to be assessed.     

The migration of bone marrow cells to thymic culture supernatants is inhibited by stress

The effect of immobilization stress on precursor cell migration from bone marrow to the thymus was studied in C57BL/6 mice. The in vitro migration assays, using Nuclepore chambers, showed that precursor cell migration to thymus supernatants was strongly inhibited in stressed animals. This inhibition of migration seemed to be cell-associated what can explain the thymic involution observed in mice under stress conditions. The migration of precursor cells from bone marrow may be one of the mechanisms by which the thymus gland is involuted by stress.     

Myocardial calpain 2 is inhibited by monoclonal antibodies specific for the small, noncatalytic subunit

Calpains (EC 3.4.22.17) are nonlysosomal intracellular proteinases which require calcium ion for activity. The calpains are heterodimers composed of a large catalytic subunit and a small subunit which may have a regulatory function during the catalytic cycle. However, whether calpains remain in the dimeric form or dissociate upon exposure to calcium is controversial. To resolve this issue, two monoclonal antibodies which specifically recognize the small calpain subunit were prepared using bovine calpain 2 heterodimer as the antigen. Both antibodies, designated P-1 and P-2, were capable of inhibiting bovine or canine calpain 2, and partially purified human erythrocyte calpain 1. However, neither could produce full inhibition. Further studies with P-1 and bovine calpain 2 indicated that the antibody decreased the calcium requirement for the proteinase. The Km for casein was increased and the Vmax was decreased. The addition of P-1 to the assay mixture several minutes after initiation of proteolytic activity resulted in a rapid inhibition. The P-1 antibody was also capable of decreasing the ability of the protein inhibitor of calpains (calpastatin) to inhibit bovine calpain 2. These studies indicate that the small subunit remains bound to the large subunit during catalysis and may influence its activity.     

The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Reduced expression of Ia antigens by thymic epithelial cells of aged mice

Thymic Ia antigen expression was examined with biochemical and immunohistochemical techniques. We found that expression of Ia antigens was reduced in thymic tissue of aged mice and that much of this loss was associated with cortical thymic epithelial cells. The ontologic pattern of Ia antigen expression in the thymus closely followed that observed for thymic weight, reaching maximal values at 4 wk of age and declining thereafter.     

Phylogenic distribution of human renal antigens defined by monoclonal antibodies

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.