Null cells in the mouse possess membrane immunoglobulins.

Null cells lacking typical T and B cell surface markers were isolated from the spleens of congenitally athymic mice by using either nylon wool or Sepharose macrobeads conjugated with rabbit antibody to mouse IgM to remove B lymphocytes. Although these null cells were negative for surface immunoglobulin by the criterion of immunofluorescence by using rabbit antisera to mouse heavy or light immunoglobulin chains, surface immunoglobulins were readily demonstrable by two alternative and independent techniques. First, by using chicken antibody to the (Fab')2 fragment of mouse IgG, nearly all null cells were positive for immunofluorescence. Second, immunoglobulins could be detected in lysates of null cells radioiodinated by the lactoperoxidase-catalyzed reaction. Analysis of the surface immunoglobulins of null cells by radioimmunoassay and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that they are 1) qualitatively similar to those of B cells and 2) present in amounts between 10 and 30% of those of B cells.     

Thymocyte plasma membrane of the rainbow trout, Salmo gairdneri: associated immunoglobulin and heteroantigens

Thymic lymphocytes of the rainbow trout, S. gairdneri were disrupted and a plasma membrane containing fraction isolated by differential and buoyant density centrifugation. Radioiodine introduced into the membrane by the lactoperoxidase catalyzed reaction and immunoglobulin (identified by radioimmunoassay with monoclonal antibody) both copurified in the plasma membrane fraction. Rabbit antibody raised to the plasma membrane fraction showed a strong reaction with trout lymphocytes in immunofluorescence, was mitogenic for trout lymphocytes, and recognized lymphocyte membrane heteroantigens of molecular weight greater than 70,000 in the thymus and 45,000-95,000 in the head kidney.     

Serum immunoglobulin M in Atlantic halibut (Hippoglossus hippoglossus): characterisation of the molecule and its immunoreactivity

Three preparations of purified immunoglobulin (IgM) were isolated from serum of Atlantic halibut (Hippoglossus hippoglossus) by means of three different methods, and each of the three IgM preparations was used to produce a polyclonal rabbit anti-halibut IgM antiserum. One of the IgM preparations was employed in the characterisation of halibut serum immunoglobulin. Halibut IgM was shown to consist of two subunits, compatible with heavy (mu) and light (L) chains. A single mu chain at approximately 76 kDa, and six possible molecular weight (MW) variants of L chain were found (range approximately 25 to approximately 28.5 kDa). IgM was glycosylated on the heavy chain and N-linked carbohydrate constituted approximately 10.3% (w/w) of the total MW of IgM. The dominant form of non-reduced IgM had a MW of approximately 780 kDa, suggesting a tetrameric structure. Non-reduced IgM also showed a number of minor protein bands. Based on estimated MW, the relative carbohydrate content and the reactivity with all three anti-halibut IgM antisera, mono-, di- and trimeric redox forms of IgM were identified. The three antisera were characterised as to specificity and reactivity by means of enzyme linked immuno-sorbent assay (ELISA), crossed immuno-electrophoresis (CIE), and immunoblotting methods. The antisera showed a considerable diversity in their specificity to the suggested MW variants of halibut Ig light chain. A method for immunohistochemical detection of IgM in tissue was established. Protein A or protein G affinity for the IgM was not detectable.     

Polyclonal immunoglobulin response of thymic,hepatic and splenic lymphocytes from fetal,germ-free and conventionally reared pigs to different B-cell activators

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes withNocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells. Dedicated to Professor J. Šterzl on the occasion of his 70th birthday     

Development of monoclonal IgA and an apparent IgG in a patient with macroglobulinemia: sharing of individually specific antigenic determinants among IgM, IgA, and IgG.

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.     

Isolation and identification of the major concanavalin A binding glycoproteins from murine-lymphocytes.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.     

Cell surface immunoglobulin of carp lymphocytes (Cyprinus carpio L.)

Molecular size and polypeptide chain composition of cell membrane immunoglobulin (mIg) on lymphocytes of carp were studied using lactopreoxidase-catalysed surface radioiodination and SDS-polyacrylamide gel electrophoresis. Carp lymphocytes prepared from pronephros, blood and thymus carry mIgM in relatively high quantity. That means about 5-10% of the radiolabelled macromolecular cell surface material precipitates as IgM. Cell surface IgM on carp lymphocytes is present as monomeric IgM (m.w. 220000-260000) and HL subunit (m.w. 110000). There are differences among molecular weights of mIg monomers of pronephric lymphocytes (m.w. 220000) and thymocytes (m.w. 260000), whereas blood lymphocytes show both components. Following reduction and alkylation H and L chains were observed. Additional thymocytic mIg possesses two unidentified components with m.w. 35000-40000 and 110000.     

Melanin concentrating hormone. IV. Development of a sensitive solid-phase radioimmunoassay for melanin-concentrating hormone

A two-step solid-phase radioimmunoassay for melanin-concentrating hormone (MCH) was developed for direct determination of the hormone in plasma samples. To this end, synthetic MCH was coupled to bovine thyreoglobulin and the complex was injected into rabbits. Specific antisera of high titer were obtained which did not crossreact with other hormones. The IgGs were chemically linked to immunobeads, an acrylamide/acrylic acid polymer matrix. In the first step, plasma MCH was immunoextracted by incubation of diluted plasma samples with anti-MCH immunobeads. In the second step, the washed polymer was incubated with radioiodinated MCH tracer for titration of non-occupied sites. This procedure made it possible to determine as little as 4 pg MCH per ml of plasma. Application of the radioimmunoassay to plasma levels of black or white background-adapted trout showed a marked difference in circulating MCH: while trout on a black background contained a mean value of 29 +/- 5.6 pg/ml, animals on a white background had 106 +/- 19 pg/ml. These findings strengthen the hypothesis that MCH is directly involved in the control of color change of teleost fishes. By contrast, there was no detectable salmonid MCH immunoreactivity in rat or human plasma.     

黄鳝IgM的分离纯化及兔抗黄鳝IgM抗血清的制备

目的 分离纯化黄鳝血清免疫球蛋白,制备其兔抗血清,并检测抗血清的特异性。方法 用Protein A亲和层析的方法纯化黄鳝血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价,通过western blotting检测抗血清的特异性。结果 纯化了黄鳝血清免疫球蛋白,免疫双扩散法测定兔抗黄鳝免疫球蛋白血清效价为1∶32,western blotting结果显示抗血清具有很好的特异性。结论 成功纯化了黄鳝免疫球蛋白,制备了兔抗黄鳝IgM抗血清,为建立黄鳝的血清学检测系统奠定了基础。     

Anti-theta Antisera may Contain Anti-allotype Contamination

THETA (θ) is a tissue-specific mouse allo-antigen present in the highest concentrations in thymus and brain^1–3. Anti-θ allo-antisera are produced after multiple injections of thymus cell suspensions into hosts differing at the θ locus, but not at the principal histocompatibility locus (H-2). Anti-θ antisera have been used by many investigators^3–9 to differentiate thymus derived lymphocytes from non-thymus derived lymphocytes and to describe the relative role of each class of cells in various immunological functions. Because it is possible that some thymocytes bear immunoglobulins bound to the cell surface, we tested the hypothesis that anti-θ allo-antisera contain antibodies directed against immunoglobulin allotype specificities of the donor, as well as antibodies to the donor θ allo-antigens.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Determination of IgM and IgG on lymphocyte surfaces by means of registering rosette formation with anti-immunoglobulin-coated erythocytes

To identify IgM and IgG on the surface of normal bovine B-lymphocytes, the test of antiglobulin-rosette formation (anti-Ig rosettes) was used. IgM and IgG were found to exist on separate B-lymphocyte populations. The composition of receptors on the surface of lymphocytes, forming Ig rosettes, was investigated. The distribution of these lymphocytes in bovine blood and lymphoid organs was determined. IgM and IgG were detected on the surface of thymocytes, their origin remains open to question.     

Xenopus laevis larval thymocytes do not express surface immunoglobulin

Xenopus laevis larval thymocytes and splenocytes were examined for the presence of Ig determinants by an indirect immunofluorescence technique, using rabbit antiserum to deglycosylated Xenopus immunoglobulins. Thymocytes had no detectable surface membrane Ig, while Ig determinants were identified on the surface of a large percentage of the lymphocytes from the spleen. The positive fluorescent staining that one obtains on the surface of thymocytes using antisera to intact Ig's is due to antibody molecules directed to the carbohydrate determinants of the Ig's which cross-react with thymocytes' surface carbohydrate determinants.     

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production

The major goal of this work is to establish a culture system for the growth of human B lymphocytes at the single-cell level so that the immunoglobulin secreted by the clonal progeny of that cell can be analyzed. A method which involves culturing small numbers (1–1000) of lymphocytes, which have been infected with Epstein-Barr virus (EBV) prior to plating, in round-bottom microtiter plates is described. A feeder layer of irradiated (2500 R) umbilical cord blood lymphocytes to which phytohemagglutinin has been added was found to be optimal. Culture supernatants collected from 3 to 6 weeks postinfection are assayed for the production of IgG and IgM by radioimmunoassay in order to determine the overall cloning efficiency of the system. We have shown that up to 33% of surface Ig-positive cells produce detectable clones in this system. Umbilical cord blood cells are superior to T-cell and macrophage cell lines as feeder layers. Furthermore, culture supernatants from phytohemagglutinin-stimulated umbilical cord lymphocytes do not adequately replace these cells. We also observed that while most IgM-secreting clones continued to produce immunoglobulin during the 7-week time period analyzed, the majority of IgG-secreting clones had a relatively short half-life in vitro. This culture system allows us to examine a significant proportion of the human B-cell population and carry out studies on the frequency of specific antibody- and isotype-producing clones.     

Receptors for antigen on lymphoid cells. II. The nature of the molecule responsible for plaque-forming cell adherence.

The nature of specific adherence of rat anti-TNP PFC to TNP-GRBC has been investigated with PLL-fixed antigen monolayers as cellular immunoadsorbents and as plaque indicators. The immunoglobulin nature of the molecule responsible for PFC adherence is suggested by the fact that pretreatment of the PFC with rabbit anti-rat Ig antisera, but not anti-histocompatibility antisera, inhibits adherence. Removal of the adherence capacity of early PFC with the proteolytic enzymes papain and pronase, or by "capping" with anti-Ig is followed by slow regeneration of the ability to adhere, suggesting that adherence is due to membrane rather than secreted immunoglobulin, the latter being detectable within minutes after enzyme treatment. Several time-related events relating to PFC adherence were observed. 1) Both direct and indirect PFC are capable of specific adherence; the ability to adhere, however, tends to decline with time, especially after secondary immunization. 2) Although early PFC adherence is unaffected by trypsin treatment, later populations become increasingly sensitive. 3) Pretreatment of PFC at various times after primary immunization with antisera specific for rat mu-chain indicates that IgM and possibly early IgG-secreting PFC have mu heavy chains on their surface. These data suggest that the PFC membrane is progressively changing during the maturation of the antibody response.     

Immunohistology of thymic nurse cells

The demonstration of thymic nurse cells (TNC), complexes between stromal cells and thymocytes, in cell suspensions of murine thymuses, prompted us to investigate (1) the relationship of TNC to other thymic stromal cell types defined in situ, and (2) the maturation stage of the enclosed thymocytes. To this purpose we incubated frozen sections of TNC suspensions with various monoclonal antisera directed to T cells and stromal cell types, using immunohistology. This approach enabled us to study antigen expression on the "nursing" cell itself and to analyze the phenotype of the enclosed lymphocytes in cross sections of TNC. The results show that lymphocytes enveloped by TNC express high levels of Thy-1, moderate levels of T200, and variable amounts of Lyt-1. Due to enzymatic degradation Lyt-2 expression could not be studied. The enveloped cells also bear PNA receptors, but no detectable I-A/E antigens. Expression of H-2K antigens on enclosed thymocytes varied from weak to absent. The "nursing" cells react with ER-TR4, a monoclonal antibody which detects cortical epithelial-reticular cells. In addition TNC express I-A/E and H-2K antigens. In contrast, TNC do not react with ER-TR 5 and 7, monoclonal antibodies, which detect medullary epithelial cells and reticular fibroblasts, respectively. TNC do not express the macrophage antigens Mac-1 and Mac-2. We conclude that TNC in vitro represent the in vivo association of epithelial-reticular cells with cortical thymocytes. However, the enclosed thymocytes do not constitute a phenotypically distinct subset of subcapsular or outer cortical cells.     

Human leukaemia-associated antigens expressed by acute lymphocytic leukaemias and their detection with heterologous antisera to T, B-, and non-T-non-B subtype AL blasts.

Antisera from rabbits and goats against subtypes of acute lymphocytic leukaemia (ALL with T-cell markers, ALL with B-cell markers, Non-T-non-B ALL) were tested for their specificity in complement-dependent in-vitro cytotoxicity testing. After absorption of the fivefold diluted antisera with erythrocytes and spleen cells of allogenous donors they reacted with ALL cells, but not with leukaemias of other types (AML, CLL, CML), lymphocytes of healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-stimulated lymphocytes, cord lymphocytes and bone marrow lymphocytes of patients in remission. In the reactions of the antisera against ALL cells the subtype of ALL is of major importance: Six rabbit antisera and one goat antiserum against T-subtype ALL reacted in all 19 tests with the leukaemia cells of 5 patients with T-cell ALL and in all 9 tests with thymocytes of 3 donors, but only in 14 out of 41 tests with the leukaemia cells of 14 Non-T-non-B ALL patients. One antiserum against a B-subtype ALL lysed B-cell ALL (1/1), but not T-cell ALL (0/3), Non-T-non-B-cell ALL (1/5) and thymocytes (0/2). Four antisera against Non-T-non-B-subtype ALL reacted in 22 out of 46 tests with the Non-T-non-B cells of 17 ALL patients, but did not react with the leukaemia cells of 4 children with T-cell ALL (0/16), one child with B-cell ALL (0/1) thymocytes of 2 donors (0/4). The reactions of the anti-ALL sera with fetal liver cells, complete absorbability of the antileukaemic activity of the antisera with fetal tissue and the reactions of an anti-fetal serum with ALL cells point to the existence of fetal antigen components as leukaemia-associated antigens.     

Separation and analysis of differentiating B lymphocytes from mouse spleens.

BALB/c spleen cells cultured for 72 hr in the presence of lipopolysaccharide (LPS) were separated into three different lymphoid cell populations on the basis of size by unit-gravity sedimentation through a linear albumin gradient. The small-cell region at the top of the gradient consisted of small lymphocytes positive for immunoglobulin light chains as shown by immunofluorescence. The intermediate-cell region in the middle portion of the gradient was composed of mitotic and intermitotic immunoblasts. Up to 90% of these cells possessed IgM, but only a very small amount of secreted IgM was detectable. The large-cell region at the bottom of the gradient contained mature plasma cells which secreted large amounts of IgM. No significant IgG synthesis was detected in cells throughout the gradient. Analysis of gradients by autoradiography sequentially during LPS stimulation showed that small lymphocytes are the primary cells responding to LPS. These cells synthesize DNA and then differentiate to become immunoblasts. The resulting immunoblasts then undergo several cycles of replication with a high percentage of these cells continuing the differentiation process to become mature plasma cells.     

Heterologous Antiserum to Thymus-derived Cells in the Guinea-pig

USEFUL antisera specific for thymocytes and peripheral T lymphocytes have only been widely available in the form of antisera to thymic isoantigens of the mouse¹. We describe here the preparation and properties of a heterologous antiserum to guinea-pig thymocytes which is rendered specific for T lymphocytes after absorption with a pure population of B lymphocytes. We have already described^2,3 the properties of the transplantable acute lymphatic leukaemia L₂C of inbred strain two guinea-pigs. The L₂C leukaemia cell is characterized as a B cell by the presence of surface immunoglobulin of the λ₂ class, the secretion of a small amount of λ₂ immunoglobulin and the presence of a receptor for antigen-antibody-complement (C3) complexes characteristic of the B cell population⁴. Because, as will be shown, the antiserum is specific both for thymocytes and thymus derived lymphocytes, it will be referred to as anti-thymus derived cell (anti-TDC) serum. The availability of such an antiserum for a species in which the in vivo and in vitro manifestations of delayed hyper-sensitivity are so easily demonstrated may prove to be highly advantageous. illustration

Open image in new window

     

Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the θ-antigen by non-ionic detergents

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid-urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the theta-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid-urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and mu-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3-4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid-urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The theta-alloantigen was isolated from CBA T-cells both by acid-urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the theta-positive thymoma WEHI 105. The theta-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.