Purification of the H-2Kk molecule of the murine major histocompatibility complex.

The intact H-2Kk antigen has been detergent-solubilized and purified using an immunoabsorbent column prepared from the 11-4.1 monoclonal antibody described by Oi et al. (Oi, V. T., Jones, P. P., Goding, J. Current Topics in Microbiology and Immunology (Melchers, F., Potter, M., and Warner, N. L., eds) Vol. 81, pp. 115-129, Springer-Verlag, New York). The mild conditions used for elution from the column, 0.5% deoxycholate in 10 mM Tris buffer, pH 8, with 0.14 M NaCl, result in recovery of 70 to 100% of the allogeneic serological activity. A murine lymphoma, RDM-4, was found to express high levels of H2-Kk; approximately 2 X 10(6) molecules/cell. Milligram quantities of H-2Kk can be purified readily using these cells.     

Localization of H-2Kk in developing mouse teeth using monoclonal antibody

The in situ distribution of H-2 antigens during mouse tooth morphogenesis was investigated using monoclonal antibodies to H-2Kk and indirect immunofluorescent techniques. H-2 antigens were detected in the basement membrane region of fetal molars; they were absent from both the epithelial and dental mesenchyme. H-2 antigens were not found in newborn and 4-day-old mouse molars.     

Neutrophil activation detected by monoclonal antibodies

Monoclonal antibodies have been produced against three neutrophil-associated membrane proteins (p 90, p 170, and p 70) expressed at different maturation stages of the cells. The reactivity of the antibodies against p 90 (B13.9) and p 170 (CLB-gran 10), as measured by quantitative flow cytofluorometry, increased after stimulation of the neutrophils by the calcium ionophore A23187, by phorbol myristate acetate, or by the chemoattractant formylmethionyl-leucyl-phenylalanine in combination with cytochalasin B. This increase is regulated independently of the simultaneously increased expression of the C3bi receptor, because neutrophils of a patient deficient for the C3bi receptor showed a normal increase in membrane expression of p 90 and p 170. Neutrophil cytoplasts were not inducible to increased membrane expression, suggesting that the cytoplasts lack the internal pool of these proteins. The reactivity of the antibody against p 70 (CLB-gram 5) was not affected by activation. The antibodies B13.9 and CLB-gran 10 may be useful to detect neutrophil activation.     

Basement membrane diversity detected by monoclonal antibodies

Abstract. Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent-cent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno)chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin     

Carbohydrate sequences detected by murine monoclonal antibodies

The carbohydrate sequences of cell surface glycolipids change during differentiation and oncogenic transformation. To detect these structural changes, murine monoclonal antibodies have been produced in many different laboratories. Some of these antibodies are used to distinguish various cell types such as normal and transformed cells, while others are used to analyze developmentally regulated antigens. Recently, the structures of many of these carbohydrate antigens have been determined. The availability of these well-defined monoclonal antibodies will be useful for the study of the regulation and function of glycoconjugates.     

Basement membrane diversity detected by monoclonal antibodies

Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno) chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin.     

Proteolytic modifications of the carboxyl-terminal region of H-2Kk

Conditions were established for the generation of limited proteolysis products from purified H-2Kk in high yield (greater than 70%). Chymotrypsin, trypsin, or papain treatment in buffer containing Nonidet P-40 resulted in removal of discrete segments from the H-2 heavy chain without detectable alteration of the beta 2-microglobulin. The Mr = 47,400 heavy chain was converted to products with Mr = 44,200, 42,800, or 40,600 by treatment with chymotrypsin, trypsin, or papain, respectively. Papain digestion removed both the hydrophilic carboxyl terminus and the hydrophobic regions. The size, detergent binding properties, and products resulting from subsequent papain treatment demonstrated that chymotrypsin or trypsin removed segments of the hydrophilic carboxyl-terminal region of the heavy chain while leaving the hydrophobic (membrane-spanning) and glycosylated NH2-terminal regions intact. Chymotrypsin and trypsin caused rapid and extensive degradation of the H-2Kk heavy chain when treatment was done in buffer containing deoxycholate, suggesting that the protein undergoes partial, but readily reversible, denaturation in this detergent. This may account for the elution of H-2K and D antigens from monoclonal antibody affinity columns by deoxycholate-containing buffers.     

Transformation of the estrogen receptor detected by two monoclonal antibodies

The estrogen receptor from fetal guinea-pig uterus is recognised by two monoclonal antibodies (H222 and H226) developed against the human estrogen receptor but it interacts differently with each of them. The H222 antibody, whose epitope is located in the hormone-binding domain of the receptor, shifts the sedimentation coefficient of the nonactivated oligomeric receptor in low salt sucrose gradients from 9S to 11S. When this oligomeric receptor-H222 complex is centrifuged in high salt gradients, it dissociates to an 8S monomer-H222 complex, indicating that all the estradiol-binding units present in the nonactivated receptor can bind the H222 antibody. In contrast, the H226 antibody, whose epitope is located close to the DNA-binding domain, shifts the sedimentation coefficient of the nonactivated receptor only to 9.4S and when this complex sediments in high salt gradients, it dissociates to a 7S monomer-H226 complex plus a 4.5S monomeric receptor not bound to the antibody. This observation suggests that not all the H226 epitopes are accessible in the nonactivated receptor. On the other hand, the temperature-activated receptor reacts with the H226 antibody to form two complexes sedimenting at 7S and 9S in high salt gradients. This 9S complex indicates the formation of a homodimer that binds two molecules of the H226 antibody. However, only one H222 epitope seems to be accessible in this dimeric form of the receptor, since only one 8S complex is observed when the activated receptor reacts with the H222 antibody. In addition, binding to the H222 antibody before activation prevents the dimerisation. This suggests that the H222 epitope is near or directly involved in the dimerisation domain. Interaction of the H222 and H226 antibodies with the estrogen receptor reveals modifications of its structure during activation, and consequently of the exposure of its functional domains.     

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.     

Clonal analysis of H-2Kb + TNP recognition by T cells with the use of H-2Kbm mutants and H-2Kb-specific monoclonal antibodies

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.     

Detection of shared H-2Kk and H-2Dk antigens that function as self determinants for cell-mediated lympholysis to trinitrophenyl-self

Spleen cells from C3H/He mice immunized in vivo to trinitrophenyl (TNP)-self were sensitized in vitro to TNP-self. These spleen cells displayed strong lysis on TNP-modified H-2D end-matched (Kd-Dk) targets as well as enhanced cytotoxicity against H-2 matched (Kk-Dk) or H-2K end-matched (Kk-Dd) target cells. Cold target-blocking studies showed that the lysis of TNP-Kd-Dk targets could be blocked by the addition of TNP-modified Kk-Dk, Kk-Dd Kk-Db, or Kd-Dk, but not by TNP-modified Kd-Dd, Kb-Db and Kq-Kq spleen cells. These results demonstrate that the lysis of TNP-Kd-Dk targets is not due to cross-reactive clones against TNP-Kd-Dd, Kb-Db or Kq-Dq antigens. Inhibition of the TNP-Kd-Dk target lysis by TNP-Kk-matched (Kk-Dd or Kk-Db) as well as TNP-Dk-matched (Kd-Dk) blockers also reveals that this target is lysed by clones directed against shared antigens between Kk-TNP and Dk-TNP, indicating that no cytotoxic response restricted for Dk-TNP only could be detected even after in vivo priming.     

Serological analysis of H-2 and Ia molecules with monoclonal antibodies

Five H-2 and seven Ia monoclonal antibodies were tested against a panel of 43 independentH-2 haplotypes (11 of laboratory-mouse and 32 of wild-mouse origin), 33 recombinantH-2 haplotypes, and up to 74 wild mice. All the antibodies gave negative reactions in the PVP hemagglutination tests; all, however, gave positive reaction with some members of the panel in the dye-exclusion cytotoxic test. Four of the antibodies (H-2.m2,Ia.m2, Ia.m5 and Ia.m7) reacted identically to conventional antibodies detecting determinants H-2.2., and Ia-1.2, Ia-1.15, and Ia-5.7, respectively (this statement does not apply to wild mice in which minor differences in reactivity patterns of the corresponding antibodies were found: the reproducibility of these differences, however, could not be checked by absorption). Five other antibodies (H-2.m5, H-2.m3, H-2.m4, Ia.ml, and Ia.m6) had very similar though not identical reactivity patterns to conventional antibodies detecting determinants H-2.5, H-2.11, H-2.25, Ia-1.2, and Ia-1.19, respectively. The last three monoclonal antibodies (H-2.ml, Ia.m3, and Ia.m4) had a reactivity pattern that did not match those of any known conventional antibodies. The near identity or great similarity of many monoclonal and conventional antibodies indicates that the cleanest of the conventional antisera are truly monospecific, and gives credence to the H-2 serology as defined by conventional antibodies. The serological analysis of monoclonal antibodies supports the true existence of private and public determinants, and reveals that the H-2 and Ia determinants are complex, even when the antibody is simple.     

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

In capping experiments with peripheral T lymphocytes, two anti-H-2.28 sera (AKR anti-AKR.L, anti-K^b, and C3H anti-0H.B10, k anti-b) that do not contain any Qa-2-specific antibodies are able to redistribute not only the H-2.28-positive H-2 molecules, but also Qa-2 molecules. This is due to the capacity of these sera to react with Qa-2 molecules because on cells where all known molecules of the H-2 ^d haplotype were capped (K1^d, K2^d, D^d, M^d, L^d, L2^d), both antisera still reacted when the cells came from a Qa-2 positive D^d strain (B10.A) but not when the cells were of Qa-2 negative strain (BALB/cByA). The reaction with la and non-H-2 antigens was excluded in these experiments. These data show that Qa-2 and H-2 antigens share some specificities of the H-2.28 family. Other anti-private and anti-public anti-H-2 sera failed to react with the Qa-2 molecules.     

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.     

Three distinct antigen systems on human B cell subpopulations as defined by monoclonal antibodies

Three novel antigen systems (L22, L23, and L24) expressed on human B cell subpopulations were identified by using TB1-2C3, TB1-2B3, and TB1-3C1 monoclonal antibodies, respectively. L22 was expressed on a minor subpopulation of B cells in human lymphoid tissues and in the peripheral blood. These B cells associated with L22 were resting small B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. This antigen was absent from all cultured hemopoietic cell lines including B cell-derived cell lines as well as from all human B cell malignancies, except for B cell-type chronic lymphocytic leukemia and hairy cell leukemia. L23 and L24, on the other hand, existed on approximately two-third of B cells in blood and lymphoid tissues. These L23 and L24 antigens were expressed largely on small lymphocytes located in the mantle zone of lymphoid follicles and to a lesser extent on large blastic cells within lymphoid germinal centers. L23 and L24, like L22, seem to disappear from B cells during their differentiation into antibody-secreting cells, because they were not expressed on normal and neo-plastic plasma cells. This is additionally confirmed by the observation that L23 and L24 were expressed little or not at all on pokeweed mitogen-activated and Epstein-Barr virus-transformed B cells, and were absent from some of B cell malignancies that have been thought to correspond to the later stages of B cell development. Although L23, but not L22 and L24, was faintly expressed on mature granulocytes and monocytes, none of L22, L23, or L24 existed on human thymus and T cells. Immunoprecipitation studies showed that L23 and L24 were different molecular species consisting of a single glycoprotein with m.w. of 205,000 and 145,000, respectively. L22 antigen is presently under study.     

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.