Studies of mouse immunoglobulin allotypes by reverse plaque assay: Detection of spleen cells secreting immunoglobulins with specific allotype in C57BL/6J mice

A reverse hemolytic plaque assay for the detection and enumeration of mouse spleen cells secreting immunoglobulins bearing a particular allotypic specificity is described. Sheep red blood cells (SRBC) coated with protein A or anti-mouse gamma globulin antibody were employed as indicator cells and an anti-allotype antibody was used as developer. A comparison of the efficiency of protein A, goat anti-mouse or rabbit anti-mouse gamma globulin antibody-coated SRBC as indicator cells in the plaque assay indicated that the rabbit anti-mouse gamma globulin-coated SRBC gave the best results in terms of number and morphology of the plaques. The number of indicator cells in the assay mixture also significantly affected the quality of the plaques formed. When the mouse spleen cells were assayed with the indicator cells and an anti-allotypic antibody as developer in presence of complement in a liquid medium, only those cells secreting the immunoglobulin of the given allotypic specificity formed hemolytic plaques.     

Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface. Interactions with concanavalin A and with Cytoplasmic structures

The effect of concanavalin A (Con A) on the capping of mouse lymphocyte surface immunoglobulin (surface Ig), cross-linked by rabbit anti-mouse Ig antibody, and on the capping of mouse thymocyte theta antigen, cross- linked by anti-theta alloantibody and rabbit anti-mouse Ig antibody, has been studied by immunofluorescence, using fluorescein conjugated Con A and rhodamine-conjugated anti-mouse Ig antibody, and by electron microscopy, using native or fluorescein-conjugated Con A and ferritin- conjugated anti-mouse Ig antibody. Prior incubation of the cells with Con A inhibited only partially capping os surface Ig, whereas it blocked almost completely capping of theta antigens. Both on cells with rings and on cells with caps the staining for surface Ig or theta antigen was superimposed to the staining for Con A. When Con A receptors on spleen cells were capped by Con A at concentrations of 10 mug/ml or higher, and the distribution of surface Ig was examined under noncapping conditions, all detectable surface Ig were found in the caps. As shown by electron microscopy, surface Ig remained dispersed in a layer of Con A. The ability of Con A to cap surface Ig was not altered by the presence of cohchicine or vinblastine. These results suggest that surface Ig are cross-linked by Con A to other Con A receptors. In these conditions surface Ig behave essentially as Con A receptors, as for example, in their sensitivity to cytochalasin B during inhibition or reversal of capping induced by this drug. The behavior of surface Ig parallels that of Con A receptors also in the presence of vinblastine. It is concluded that in the presence of Con A, antimitotic drugs do not modify directly the interaction between Con A receptors and surface Ig, but probably influence the capping ability of the Con A receptors or, more in general, affect the ability to elicit movements over the cell surface. The role in capping of cytochalasin- sensitive and vinblastine-sensitive structures is discussed. Both types of structures appear to play an active role in the formation of a cap, although the former probably corresponds to the main mechanical system responsible for the active displacement of cytoplasmic and surface material.     

Fate of surface immunoglobulin during induction of lymphocyte proliferation

The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose-linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose-linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose-linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose-linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are considered.     

Anti-immunoglobulin-induced proliferation of B cells : Parallelism in the inhibition by chloroquine, monensin and immunoglobulin

Proliferation of rabbit lymphocytes was induced with goat anti-rabbit immunoglobulin. Chloroquine and monensin, known to inhibit internalization-related events, yielded inhibition of proliferation that paralleled the inhibition by a specific competitive ligand, rabbit immunoglobulin (IgG), whereas inhibition by puromycin did not. Moreover, virtually all of the cells that can be activated in freshly isolated populations adhered to anti-immunoglobulin-coated Petri plates, whereas all of the activatable population was recovered in the non-adherent fraction after a brief incubation of the cells with anti-immunoglobulin to induce internalization of surface membrane immunoglobulin. Using immunofluorescence it was further observed that monensin and Chloroquine inhibit the reappearance of surface immunoglobulins on the cell surface to some extent subsequent to their removal induced by anti-immunoglobulin.     

Effect of molar ratio of fluorescent dye to IgG on the detection of lymphocyte surface immunoglobulins by immunofluorescence

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S-Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained. 1. The percentage of S-Ig bearing cells increased markedly when labeled anti-human H- or L-chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S-Ig was caused by increased non-specific adsorption of the fraction of labeled antibody with a high F/P molar ratio. 2. This non-specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mcie showed less non-specific adsorption than those from humans. 3. It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.     

Immunoglobulin secretion by human splenic lymphocytes in vitro: the effects of antibodies to IgM and IgD.

The effects of F(ab')2 fragments of affinity-purified rabbit anti-human mu chain antibody (RaHmu) and rabbit anti-human delta chain antibody (RaHdelta) on spontaneous and mitogen-stimulated immunoglobulin (Ig) secretion by normal human spleen cells were studied. IgM and IgG secretion by human spleen cells cultured in vitro was measured by incubating the cells with 3H-amino acids precipitating the secreted labeled Ig with anti-Ig, and analyzing the precipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both RaHmu and RaHdelta suppressed spontaneous and LPS-induced IgM and IgG secretion as well as PWM-stimulated IgG secretion. In different experiments, RaHmu and RaHdelta either suppressed or augmented PWM-induced IgM secretion. The anti-Ig induced augmentation of PWM-triggered IgM secretion was most apparent when spleen cells were cultured at lower cell densities or when lower concentrations of anti-Ig were employed. These date indicate that perturbation of B cell surface immunoglobulin receptors with specific anti-Ig antibody can alter markedly the ability of these cells to differentiate into antibody-secreting cells.     

Preparation of liposomes possessing immunologic specificity]

The authors obtained artificial lipid vesicles--liposomes containing immunoglobulins. IgG in the complexes with liposomes proved to retain their immunological activity: the liposomes containing rabbit anti-mouse IgG agglutinated in the presence of donkey anti-rabbit IgG or mouse serum. As shown by the use of liposomes containing H3-inulin and immunoglobulins against the cell surface determinants, these immuno-liposomes selectively bound the target, but not the control cells. Specific binding with the antigenic cell surface determinants was also demonstrated in the case of liposomes bearing the nonimmune globulins besides the immunoglobulins. By the indirect immunofluorescence method it was shown that the nonimmune globulins in complex with the immune liposomes were selectively bound by target cells. A possible use of the immuno-liposomes to deliver various substances selectively to the cells of particular types, and to incorporate new antigens into the cell membrane is discussed.     

Schistosoma mansoni: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

Selective Cytotoxicity of Anti-Kappa Serum for B Lymphocytes

MOUSE lymphocytes can be divided into two distinct populations according to the density of immunoglobulin determinants on their surface. Lymphocytes with a high density of immunoglobulin are marrow-derived, nonthymus-processed, B cells, whereas lymphocytes with little or no immunoglobulin are thymus-derived, T cells^1–3. Since more than 95% of mouse immunoglobulin light chains are of the kappa type⁴, treatment of lymphocyte suspensions with an appropriate dilution of rabbit anti-mouse kappa serum and complement should be cytotoxic for only B lymphocytes. This prediction was tested by using lymphocyte populations enriched for either T or B cells or containing the two cell types in a known proportion.     

Amino acid sequence of the N-terminal sixty-nine residues of heavy chain derived from a homogeneous rabbit antibody

The sequence of the N-terminal 69 residues of heavy chain from a homogeneous rabbit antibody to type III pneumococcal polysaccharide was determined. The sequence is similar to that found in heavy chains of normal pooled rabbit immunoglobulins of the same allotype Aa1. Two regions of the homogeneous heavy chain (residues 35-46 and 62-69) are very similar to corresponding regions of heavy chains from rabbit Aa2 immunoglobulin, as well as from mouse, guinea-pig and human immunoglobulins. In contrast, residues 47-62 appear to be variable. Comparison in this section with another homogeneous anti-pneumococcal antibody (Strosberg et al., 1972) of related specificity and of the same allotype indicates sequence variation in at least three positions. An antibody to group C streptococcal carbohydrate of allotype Aa2 (Fleischman, 1971) differs by five amino acids in the same region of the heavy chain. Sequence variability between these three antibodies does not occur in homologous positions within this variable section. Allotype-related sequences could not be identified in section 34-65.     

Selective elimination of a B cell subset having acceptor site(s) for T cell-replacing factor (TRF) with biotinylated antibody to the acceptor site(s) and avidin-ricin A-chain conjugate

A covalent conjugate of avidin with ricin subunit A-chain (avidin-RA) was prepared by using N-succinimidyl 3-(2-pyridyldithio)propionate as a coupling agent. Selective cytotoxic activity after the combined treatment of spleen cells with biotinylated antibody and avidin-RA was demonstrated by the fact that the responsiveness to LPS was selectively abrogated by pretreatment of the cells with biotinylated rabbit anti-mouse immunoglobulin (MIg) antibody, but not with biotinylated anti-Thy-1.2 antibody. Neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the responses to mitogens. Moreover, a high anti-DNP PFC response elicited by DNP-KLH-primed BALB/c mouse spleen cells stimulated in vitro with DNP-KLH was mostly abrogated by the pretreatment of the cells with biotinylated anti-MIg antibody and avidin-RA. Again, neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the anti-DNP PFC response. This cell-killing method with the use of biotinylated antibody and avidin-RA was applied and evaluated in experimental systems in which the helper action of T cells on B cells was mediated by T cell-replacing factor (TRF) or was performed by the direct interaction of T cells with B cells (cognate interaction). When DNP-KLH-primed splenic B cells, pretreated with biotinylated F(ab')2 fragment of DCF1 male anti-BALB/c-B IgG antibody against acceptor site(s) for TRF followed by treatment with avidin-RA, were stimulated with DNP-OVA in the presence of monoclonal TRF, the anti-DNP PFC response was significantly decreased, whereas the same treated B cells responded well to stimulation with DNP-PPD in the presence of Tbc-primed T cells (cognate interaction). These results indicate that B cells responsible for the cognate interaction and those having TRF acceptor site(s) belong to a distinct subpopulation of B cells, and that the cytocidal action of the noncovalent conjugate of the antibody and RA formed from the biotinylated antibody and avidin-RA via an avidin-biotin complex has immunologic selectivity, eliminating only the latter subset of B cells recognized by the antibody.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. II. Sustained antigen-specific T cell help is required to induce a specific antibody response.

The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Immunoglobulin production by a human-mouse somatic cell hybrid

The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobulin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.     

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.     

Expression of an unidentified immunoglobulin isotype on rabbit Ig-bearing lymphocytes.

A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.     

Immunohistochemical localization of basic fibroblast growth factor within the mouse uterus.

Uterine samples were either rapidly frozen in liquid nitrogen or placed in Bouin's fixative. A commercial primary polyclonal antibody made in rabbits against human recombinant basic fibroblast growth factor (bFGF) was used. Western blot analysis indicated that the antibody was specific for bFGF and did not react with acidic FGF. The primary antibody was followed by either goat anti-rabbit immunoglobulin G (IgG) conjugated to the fluorescent phycobiliprotein tracer phycoerythrin or biotinylated goat anti-rabbit IgG and a biotin-avidin-peroxidase complex. Specificity controls using adjacent sections were carried out by (i) substituting normal rabbit sera for the primary antisera, (ii) omitting the primary antisera or (iii) extracting sections with NaCl (2 mol l-1) prior to the immunochemical procedures. No binding of the antibody was observed with any of the specificity control sections. The connective tissue stroma and the basal lamina associated with uterine glandular and surface epithelial layers were positive for bFGF. Localization was not observed within surface or glandular epithelial cells. The basal lamina and endothelial cells associated with blood vessels within the uterus and the smooth muscle cells of the myometrium were positive for bFGF. There were no differences in uterine localization patterns or intensity during the oestrous cycle or after ovariectomy and steroid hormone supplementation. These studies demonstrate the specific localization of bFGF within the mouse uterus.     

Anti-carbohydrate activity of T cell-reactive chicken anti-mouse immunoglobulin antibodies

The specificity of chicken anti-mouse immunoglobulin F(ab')2 antibodies, which bind readily to T cells and have been claimed to detect immunoglobulin on T cells, was analyzed by indirect immunofluorescence. It ws found that only a minor proportion of the total antibody bound to T cells whereas the major fraction bound only to B cells. The T cell-reactive antibody cross-reacted with bovine IgG and therefore could be purified on this basis by affinity chromatography. T cell staining was inhibitable with nonimmunoglobulin glycoproteins as well as immunoglobulin, in contrast to staining with the B cell-specific antibody fraction, which was only inhibited by immunoglobulin. This suggested that the T cell staining was caused by anti-carbohydrate antibody. Inhibition of T cell staining was abolished by periodate oxidation of the inhibitors but not by proteolytic digestion. In addition, the antigenic determinant on the cell surface was not destroyed by autoclaving. Finally, antibody was absorbed on a mucin-Sepharose column and anti-T cell activity was found in the bound but not the unbound fraction. These results strongly suggest that the only anti-T cell activity in the chicken antibody is anti-carbohydrate antibody.