A new mouse cell-surface antigen (Ly-m18) defined by a monoclonal antibody

Spleen cells from C3H/An mice immunized with spleen cells of C57BL/6-H-2 ^k mice were fused with myeloma cell line NS.1. One established hybrid cell line continuously secreted antibody that recognized a new surface antigen provisionally called Ly-m18. The new alloantigen is expressed on 90 percent of thymus cells, 55 percent of spleen cells, and 45 percent of either lymphnode or bone-marrow cells. It is also expressed on cells derived from brain, kidney, and liver. Fifty percent of either peripheral T or B cells express the Ly-m18 antigen, and some tumor cell lines with T, B, pre-B or stem cell characteristics are Ly-m18 (+). The strain distribution pattern distinguishes Ly-m1 8 antigen from all other murine lymphocyte alloantigens. The typing data of two sets of CXB and AKXL recombinant inbred strains indicate that the Ly-m18 gene is linked to the Ltw-2 locus which has not yet been assigned to a chromosome.Abbreviations used in this paper RI recombinant inbred - Con-A concanavalin A - LPS lipopolysaccharide - MLR mixed lymphocyte reaction The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

Composition of the immune system of allophenic mice.

Mice were immunized with antigen (Rabbit Fab' fragments) attached to syngeneic, or f₁ (semi-syngeneic) irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers showed that the response towards antigen on syngeneic or F₁ cells, was significantly lower than that towards the same antigen on allogeneic cells. By subsequent in vitro incubation of immune spleen cells with antigen followed by plaque assay, it was found that those spleen cells exhibiting lowered plaque forming cell numbers initially, (i.e., those mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which initially (before in vitro incubation) demonstrated a larger response (i.e. those mice immunized with antigen on allogeneic cells).     

Adjuvant Activity of Mycobacterial Fractions

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using ⁵¹Cr-labeled target cells. C57BL/6J mice (H-2^b) were immunized intraperitoneally with mastocytoma cells (H-2^d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 × 10⁷), could be induced in spleens of mice immunized with a mixture of mastocytoma cells (10⁴) and oil-attached BCG-CWS. The enhancing effect persisted for 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10⁴) plus oil-attached BCG-CWS did not increase to the maximum level as was observed in mice immunized with a larger number of mastocytoma cells (3 × 10⁷) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 × 10⁷) alone.     

Host defense in cryptococcosis. III. In vivo alteration of immunity

The present studies utilize an inbred mouse model to evaluate the adoptive transfer of spleen cells to augment immunity against Cryptococcus neogormans. Protection against cryptococcosis was transferred using spleen cells obtained from mice surviving cryptococcosis. These spleen cell donors had no detectable anticryptococcal antibody. Also, treatment with antimouse-thymocyte globulin ablated dermal hypersensitivity reactions of immunized mice, and shortened survival in both immunized and unimmunized mice. These in vivo studies further support a major role for cell-mediated immunity in host defense against experimental cryptococcosis.     

Monoclonal antibodies againstAgrobacterium tumefaciens strain C58

Hybridoma cell lines were derived from the fusion of mouse myeloma cells with spleen cells from a mouse that had been immunized withAgrobacterium tumefaciens strain A759, a flagella-less mutant of strain C58 containing the Ti plasmid of strain B6. All of the 20 antibodies produced by the cloned hybridomas reacted with strain C58 and with other strains derived from C58. The antibodies did not react with 34 other strains ofA. tumefaciens, representing the three biovars, or with strains ofA. radiobacter, A. rubi, Rhizobium leguminosarum, R. meliloti, or other plant-associated bacteria such asErwinia herbicola andPseudomonas syringae. In addition to reacting with whole cells of strain A759, the antibodies reacted with phenol-water extracts of A759, indicating that they may recognize the lipopolysaccharide. These antibodies may be useful for ecological and epidemiological studies ofA. tumefaciens strain C58 in the agroecosystem.     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

The histophysiology of antibody-forming sites in the marine toad

Summary The normal histologioal features of the lymphatic organs — pericardial nodes, jugular bodies, spleen, and kidney — of the marine toad, Bufo marinus, are decribed. The lymph nodes and spleen of the marine toad lack the compartmental organization of corresponding mammalian organs and contain relatively less internal connective tissue. The cellular stroma composed of reticulum cells and fixed macrophages plays a more important role in maintaining structural organization than do the connective tissue.Changes in the cellular composition of the lymphatic parenchyma were observed in animals immunized with bovine serum albumin suspended in Freund's complete adjuvant. In addition to an increase in the number of lymphocytes and the presence of lymphoid hemocytoblasts, cells occurred which possessed many of the morphological characteristics of mammalian plasma cells. These plasma cells, which exhibited positive fluorescent antibody reaction, were more abundant in the kidney than in the lymph nodes or spleen of an immunized animal.Granulomas developed at the site (gastrocnemius muscle) of injection of antigen in complete adjuvant, and similar cystic lesions arose in the kidney. Apparently, the antigen-adjuvant mixture found its way from the site of injection (gastrocnemius muscle) into the kidney, probably via the renal portal system, and established lesions in the kidney. Appreciable numbers of antibody-forming cells, or plasma cells, were found in the muscle granulomas and in the kidney lesions.The lymphoid tissue of the kidney is considered the principal site of antibody formation in the marine toad, Bufo marinus.This investigation was supported by grants HD-2614-1 and GM-11782 from the United States Public Health Service administered by Dr. Ronald R. Cowden and Dr. E. Peter Volpe, respectively.     

The wall associated lipoteichoic acid ofStreptococcus sanguis

A competitive ELISA is described for the measurement of lipoteichoic acid. The assay was used to determine the wall associated lipoteichoic acid ofStreptococcus sanguis which was found to represent only 2–4% of the phenol extractable content. Extracellular lipoteichoic acid was detected even after exhaustive cell washing. This material was not the result ofde novo synthesis because membrane de-polarization had no effect on the amount detected. Since extracellular lipoteichoic acid interfered with the measurement of cell surface antigen, cells were fixed with glutaraldehyde prior to assay. Lipoteichoic acid was demonstrated on the surface of fixed cells which did not leak antigen. The relevance of fixation used in antigen location studies by electron microscopy of immune-labelled cells is discussed.     

Protection against dysentery infection (Shigella sonnei) by cells of peritoneal exudate,spleen, thymus,bone marrow and mesenteric lymph nodes of non-immune and specifically immunized mice

Using wild white mice both as donors and recipients, transfer of cells from peritoneal exudate, spleen, thymus, bone marrow and mesenteric lymph nodes were performed. The cells were isolated from non-immune or immunized mice. Mice were immunized either with living dysenteric bacteria or with various preparations obtained from these bacteria. The protective activity of transferred cells was studied in recipients infected with a lethal dose of a living culture ofShigella sonnei.     

Immunosuppression in experimental cryptococcosis in rats

Using a rat model, we have previously demonstrated that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a non-related antigen, human serum albumin (HSA), that has been injected 7 days after the infection. We previously determined that the cryptococcal infection induces afferent suppressor or suppressor induction cells (Ts₁) to HSA. The primary objective of the present study was to investigate the suppressor cells involved in the efferent phase of delayed-type hypersensitivity (DTH) response to HSA in rats infected with C. neoformans and immunized with the non-related antigen and determine the role that the Ts₁ cell plays in the induction of that cell. For this purpose, the spleen mononuclear (SpM) cells containing the Ts₁ or SpM cells from immunized non-infected rats (used as donor controls) were transferred to two groups of syngeneic naive recipients (first recipients). Later, the SpM cells from both groups of animals were transferred to rats immunized with HSA (second recipients). The efferent limb of the DTH response to HSA was suppressed in the recipients that received SpM cells from donors injected with Ts₁ cells. Additional HSA antigen was not required for induction of these efferent suppressor cells. Furthermore, we here show that these cells are resistant to treatment with cyclophosphamide (Cy), and that they can activate another suppressor population. The latter are Cy sensitive and are present in the immune recipient.     

Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensions against DBA mastocytoma cells

Spleen cell populations stimulated in vitro with as few as 1000 tumor cells produce cytotoxic effector cells. Syngeneic as well as allogeneic spleen cells respond to DBA mastocytoma tumor cells. There is a significant cellular immune response to allogeneic tumor cells 72 hr after exposure to antigen. By contrast, the response of DBA spleen cells to DBA mastocytoma tumor cells is first detectable at 120 hr following exposure to antigen. C57BL/6 spleen cells immunized against DBA mastocytoma antigen kill both DBA mastocytoma tumor cells and normal cells from DBA animals. DBA spleen cells immunized against DBA mastocytoma antigen kill only the DBA mastocytoma tumor cells, and not normal cells from DBA animals.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus]

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.     

Transfer Agent of Immunity

Antibody formation in vitro was studied using erythrocytes (RBC) as antigen and immunocytoadhesion as the technique for detection of antibody-forming cells. Spleen cells (SPC) of nonimmune mice gained the ability to produce antibody after treatment with ribonucleic acid (RNA) preparation extracted from allogeneic mice immunized with xenogeneic or allogeneic RBC. It was also found that a small proportion of SPC from individual mice of certain strains formed antibody against autologous RBC when the cells were treated in vitro with RNA preparation obtained from the spleen of an allogeneic mouse immunized with RBC of that individual. No converting ability was observed in the RNA preparation from spleen of nonimmune autologous or allogeneic mice. The converting activity of immune RNA preparation was shown to be sensitive to ribonuclease treatment. These evidences exclude the possible contribution of antigen or fragments thereof in the RNA preparation to the induction of antibody formation in RNA recipient cells.     

Novel heterophile chicken antigen: immunohistochemical localization using antisera to Mycobacterium smegmatis and possible association with lymphocyte maturation

Summary A novel heterophile antigen shared byMycobacterium smegmatis and chicken tissues was demonstrated by the indirect immunoperoxidase method using antisera raised in rabbits immunized with a complete Freund's adjuvant containing killedMycobacterium smegmatis as an immunostimulating component. This antigen was strongly expressed in medullary lymphocytes of the thymus and bursa of Fabricius, but was undetectable in lymphoid cells of the cortical regions of these organs. Only a few lymphocytes stained positively for the antigen in T- and B-cell areas of the spleen. These data suggest that the heterophile antigen is associated with the intrathymic and intrabursal maturation of chicken lymphocytes. The antigen was also detected in some nonlymphoid cells. It was not found in sheep erythrocytes, human and rat tissues or in killed bacillus Culmette—Guerin.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

The conveyer hypothesis is based on the fact that because of clone predetermination, antibody production takes place in an organism without the presence of antigen as a result of natural cell differentiation. Soluble antigen is an analogue of a specific mitogen which gives rise to reproduction mainly of cells carrying on their surface the immunoglobulin receptors to the given antigen. The mathematical model of the conveyer hypothesis takes into account the initial conditions, among them the background level of antibody-producing cells before injection of a soluble antigen, migration of precursor cells in the draining lymphoid organ, and the rate of precursor differentiation, including the rate of the change of the immunoglobulin receptor number on the cell surface. Changes of antigen concentration in blood determine the intensity of precursor proliferation. Comparison of real experiments (intraperitoneal injection of capsular antigen ofPasteurella pestis into inbred mice) with calculations done on the basis of the developed mathematical model shows a definite qualitative resemblance with the kinetics of antibody-producing cells and free antibodies as well as with the decrease of free antigen concentration in blood. In spite of some differences between model experiments and real experiments the conveyer hypothesis and its mathematical model appear suitable for describing the primary immune response of mice immunized with low doses of capsular antigen ofPasteurella pestis.     

In vitro screening of seaweed extract on the proliferation of mouse spleen and thymus cell

A total number of 31 types of seaweed were assessed with regard to their effects on the proliferation of mouse spleen and thymus cells in a culture, using an MTT reduction assay. Acetone:dichloromethane (1∶1) extracts of three seaweed plants:Derbesia marina, Sargassum sp., andHisikia fuziformis, exhibited significantly positive effects on the survival of mouse spleen and thymus cellsin vitro. The acetone: dichloromethane (1∶1) extracts ofSargassum sp., in particular, much more potent effects on thymus cell activation than did any of the other types of seaweed. However, the methanol extracts ofSargassum ringgoldianium andChondrus crispus exerted a stimulatory influence only on the proliferation of mouse spleen cells, whereas the methanol extracts ofGrateloupia lanceolata exhibited significant cell proliferation properties in both spleen and thymus cells.     

Ly-m11: TheH-3 region of mouse chromosome 2 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with B10.S spleen cells were fused with the nonsecretor myeloma line NS.1. One established hybrid cell line continuously secreted antibody that recognized a new antigenic specificity, tentatively called Ly-mll. This newly found antigen is detectable on nearly 100 percent of spleen and lymph-node cells, 70 percent of bone-marrow cells, and 20 percent of thymus cells by direct cytotoxicity assays, and on the cells derived from kidney and liver. Strains that are Ly-mll (+) include C57BL/6, C57BL/10J, B10.S, C57BR/cdJ, C57L/J, and C57BL/KsJ. Other mouse strains so far tested are Ly-m11 (–). The strain distribution pattern distinguished Ly-mll from any known murine lymphocyte alloantigens, but it follows theH-3 ^a haplotype which is defined by skin transplantation. Linkage tests of nine congenic strains ofH-3 and/orH-13/a loci and five recombinant inbred lines including CXB, BXH, AKXL, SWXL, and BXD revealed no recombinations betweensH-3 andLy-m11 loci on chromosome 2. This newly discovered Ly-m11 alloantigen could itself constitute a minor histocompatibility antigen detectable by serological means.Abbreviations used in this paper RI recombinant inbred - H histocompatibility - a non-agouti - B10 C57BL/10Sn The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

细胞内POCR法克隆抗体可变区基因

报道一种克隆免疫脾细胞中抗体可变区基因的新方法。抗原免疫小鼠的脾细胞经分散、固定、渗透处理,直接在细胞内进行反转录,再采用半巢式PCR扩增,获得的序列经测序证明是抗体可变区编码序列。这种不经mRNA纯化、从细胞内直接获取目的的方法既可用于抗体库的建立,亦可用于新基因的快速克隆。