The production of immunoglobulins by human peripheral blood lymphocytes in vitro

We have demonstrated that the peripheral blood lymphocytes of normal humans show an increased production of immunoglobulins in vitro after non-specific stimulation by phytohaemagglutinin (PHA). The methods used include immunofluorescence and immune co-precipitation. Specific antigens to which the donor has been sensitized produce a similar effect in a smaller number of cells. The cells of persons with defects of immunoglobulin production can be stimulated by PHA to enlarge and divide, but their immunoglobulin content is abnormal and reflects the pattern of the circulating serum globulins. Preliminary results suggest that a portion of the immunoglobulin produced after either specific or non-specific stimulation of cells from sensitized donors behaves like specific antibody.     

Sheep, rabbit and chicken antisera against a human VH fragment: reactivity with immunoglobulins and lymphocytes

Antisera against a human VH fragment obtained from an IgG3, VH II, kappa protein (KUP) were raised in rabbits, sheep and chicken. The three types of anti-VH antisera reacted equally well with both intact immunoglobulin molecules and isolated heavy chains. The antisera did not detect any free heavy chain specific antigens by sensitive enzyme-linked immunosorbent assay (ELISA) tests although they reacted with some antigens which were more or less hidden on intact immunoglobulin molecules but well expressed on isolated heavy chains. The antisera reacted with more than 90% of IgG, IgA and IgM present in normal pooled serum. Experiments with T cells from normal peripheral blood indicated that the sheep and rabbit anti-VH antisera reacted with a 70,000 mol.wt T-cell surface antigen.     

A test for antigen--antibody complexes in human sera using IgM of rabbit antisera to human immunoglobulins.

A simple semi-quantitative test for soluble antigen--antibody complexes using characterized non-human reagents and permitting analysis of their constituents is described. The agglutination of immunoglobulin-coated latex particles by rabbit IgM antibodies to the immunoglobulin is inhibited by complex-containing sera. No inhibition is obtained with monomer immunoglobulins. Semi-quantitative measurement of complexes may be made by electronically counting residual unagglutinated latex particles. The new method of linking proteins to latex by DNP coupling permits the technique to be applied to all constituents of complexes; immunoglobulins, complement and antigens. A new method of decomplementing sera by EDTA-Sigma cell-IgG absorption allows analysis of sera without the false positives and false negatives other methods give. The test gave positive results for IgG complexes in most of twenty-four patients with SLE nephritis. IgA complexes were identified in a patient with Henoch-Schonlein purpura nephritis.     

Membrane-associated immunoglobulins of human lymphocytes in immunologic disorders

Membrane-associated immunoglobulins of peripheral blood lymphocytes were studied by indirect immunofluorescence for γ, α, μ, κ and λ chains in healthy subjects and patients with immunologic disease.

In healthy subjects, heavy chains were found on 30·7% of lymphocytes (γ 15·3%, α 7·2% and μ 8·2%) and light chains on 32·8% of cells (κ 20·4% and λ 12·4%). Patients with humoral immune deficiencies had fewer immunoglobulin-bearing cells; sarcoidosis or thymectomy patients had normal or decreased immunoglobulin-bearing lymphocytes; cells with light chains were fewer than those with heavy chains on their lymphocytes. In some cases, normal levels of serum immunoglobulins were found in the absence of the corresponding immunoglobulin-bearing cells, and in others normal immunoglobulin-bearing lymphocytes were present in the absence of the corresponding serum immunoglobulins.

These data suggest that (1) immunoglobulin-bearing lymphocytes in blood do not reflect the condition of immunoglobulin-synthesizing cells in peripheral lymphoid tissues, and (2) in certain immunologic disorders, either some B-lymphocytes do not synthesize immunoglobulins, or immunoglobulins are in such a situation that the whole molecule or part of the molecule is not visualized by current methods.

     

Complement-dependent cytolysis of human B lymphocytes with anti-light chain antisera

Using anti-light chain antiserum and rabbit complement, 12 % of human peripheral blood lymphocytes and 42 % of tonsillar lymphocytes were specifically lysed. From control experiments it could be assumed that these cells are B lymphocytes. Under comparable conditions, cytolysis of tonsillar lymphocytes on a preparative scale was achieved. After removal of the lysed cells by Ficoll-Isopaque centrifugation, the T/B cell compositions of the remaining living cell population and the untreated cell suspension were compared using the immunofluorescence technique for demonstration of surface immunoglobulin-bearing cells and the rosette tests for sheep erythrocyte-binding and complement-receptor leukocytes. A maximal B cell depletion of 97 % and a T cell enrichment up to 139 % could be obtained. The applicability of the described method for studies on lymphocyte functions is discussed.     

Induction of protein disulphide-isomerase and immunoglobulins by pokeweed mitogen in human lymphocytes.

The Protein disulphide-isomerase (PDI, EC 5.3.4.1, Thiol-proteindisulphide oxidoreductase, EC 1.8.4.2) is thought to regulate the sulfhydryl status of cells and to catalyze thiol/disulphide exchange reactions involved in the post-translational processing of disulphide containing secretory proteins. The aim of the present investigations was to study the possible function of this enzyme in differentiation of B lymphocytes and immunoglobulin synthesis. Non-adherent human mononuclear cells or purified T cells were cultured in presence and absence of Pokeweed mitogen over 3, 5 and 7 days. Monoclonal antibodies and a rabbit polyclonal antiserum specific for human liver PDI were produced to determine the concentration of PDI by an ELISA technique and cytoplasmic immunofluorescence. After PWM stimulation, both, the cellular content of PDI as well as that of immunoglobulin, particularly IgM, have been found to be induced in a time dependent manner with a 2-3 fold increase in comparison to unstimulated cells. The specific induction of PDI in human B lymphocytes was also confirmed in Western blotting. Our findings suggest that PDI plays a critical role in the final stages of B cell differentiation and immunoglobulin synthesis by activated B cells and plasma cells, respectively.     

The cytotoxic effect of heterologous antisera to mouse IgG on mouse lymphocytes

Goat and rabbit anti-mouse IgG were significantly cytotoxic for adult mouse lymphocytes from spleen, lymph node, peripheral blood, and to a lesser extent, bone marrow. Newborn mouse spleen lymphocytes were not susceptible to this cytotoxic effect, but spleen lymphocytes from 1 week old and 1 month old donors were increasingly so. Absorption of anti-mouse IgG sera with mouse spleen cells or mouse IgG removed both IgG precipitins and lymphocytotoxins. Absorption with mouse red cells or foetal tissue, however, did not remove either cytotoxins or IgG precipitins. Antisera directed against human or rabbit IgG's were not cytotoxic for human or rabbit peripheral blood lymphocytes.     

Analysis of the transformation of human lymphocytes by Epstein-Barr virus III

T-cell depleted human cord blood lymphocytes were exposed to P3HR-1 strain of Epstein-Barr virus (EBV) and simultaneous observations of immunofluorescence, cellular morphology, and autoradiography were carried out in individual cells. Soon after infection, nuclear antigen (EBNA) synthesis, blastogenesis, and DNA synthesis occurred, as was previously observed in B95-8 strain EBV infection. Although mitosis followed with characteristic cell aggregate formation, the cell proliferation was temporary and death followed in about 2 weeks. The synthesis of the early antigens (EA) and the viral capsid antigen (VCA) were not significant. These findings seem to indicate that the strain P3HR-1 EBV is capable of inducing early events of transformation in primary human B-lymphocytes, but the cells infected in this way have a short life span.     

Serological precipitation method for studying biosynthesis and secretion of immunoglobulins by human peripheral blood lymphocytes

A reproducible, serological method to quantify radiolabeled immunoglobulin (Ig) synthesized and secreted by human peripheral blood lymphocytes is described. In contrast to indirect precipitation (rabbit antihuman Ig and goat antirabbit Ig), the direct coprecipitation method using human IgG and rabbit antihuman IgG F(ab)₂ showed much smaller, non-specifically precipitable radioactivity (i.e., 2～14%) without affecting Ig radioactivity.     

Study of human T and B lymphocytes with heterologous antisera. I Preparation, specificity and properties of antisera.

Heterologous antisera have been raised in the horse and rabbit against human lymphocytes. Appropriate absorptions on either B or T cells were performed to make antisera specific for human T (anti-HTLA) or B (serum 789) lymphocytes respectively. In addition serum 789 was found to react with circulating monocytes. The percentages of T and B cells detected by anti-HTLA and 789 sera in the different lymphoid organs averaged respectively: 78-7 per cent and 14-7 per cent in peripheral blood, 91-4 per cent and 4-0 per cent in thymus, 73-0 per cent and 14-5 per cent in lymph nodes, 53-6 per cent and 30-0 per cent in spleen, 47-1 per cent and 47-6 per cent in tonsils and 17-5 per cent and 13-5 per cent in bone marrow. Anti-HTLA serum appeared to supress E-rosette formation but did not affect binding of C3-coated erythrocytes. Serum 789 did not prevent E-rosette formation and reduced the number of EAC rosettes by 50 per cent. Anti-HTLA serum was found able to suti-lymphocyte serum, and PWM in the presence of complement; it was found highly mitogenic by itself. Serum 789 decreased the proliferative response to phytomitogens in about the proportion of cells killed by the antiserum. These results indicate that the presence of T cells is necessary for the mitogen-induced proliferation to occur, and that B lymphocytes are induced to proliferate in the presence of T cells and phytomitogens. Anti-HTLA serum was found not to inhibit K-cell activity of lymphocytes against antibody-coated chicken erythrocytes. These antisera appear very useful tools for the study of the role of human B and T lymphocytes involved in in vitro immune reactions.     

The formation of immunoglobulins by human tissues in vitro: IV. Circulating lymphocytes in normal and pathological conditions

The formation of immunoglobulins by circulating lymphocytes was studied by three techniques: (1) Autoradiographic analysis of the immunoglobulins synthesized during the incubation of cell suspensions in a medium with radioactive amino acids; (2) direct immunofluorescent staining; and (3) examination of the cellular morphology.

Lymphocytes of the normal peripheral blood were found to synthesize a distinct amount of IgG and smaller amounts of IgA and IgM. Cells of the thoracic-duct lymph synthesized distinct amounts of all three immunoglobulins. A similar pattern was found in infectious mononucleosis and rubella. In infectious mononucleosis the significantly increased synthesis of IgM during the first 10 days of illness led to the supposition that this result may be due to primary antigenic stimulation. The pattern in chronic lymphatic leukaemia is characterized by the consistent absence of IgA and the labelling of IgG, mainly the medium to high mobility part, and of IgM. In agammaglobulinaemia a trace of IgG and IgA was found in one case; the other was entirely negative.

The immunofluorescent staining showed that in all samples some of the medium-sized lymphocytes contain IgG, IgA or IgM. Peripheral blood samples taken during an infectious mononucleosis or rubella infection and thoracic duct lymph revealed also positive large lymphocytes and plasma cells.

A remarkable observation was the weak fluorescence of small lymphocytes which were exclusively positive for IgM. It is postulated that these small lymphocytes indicate their initial synthesis of IgM antibodies when engaged in primary response.

     

Stimulation of lymphocytes by allogeneic lymphocytes and lymphoblasts in the presence of anti-HLA antisera.

Anti-HLA alloantisera inhibit mixed lymphocyte responses in which normal lymphocytes are used as stimulator cells. These same antisera are unable to inhibit lymphocyte proliferative responses stimulated by lymphoblastoid cells from cultured lymphoid cell lines. They also fail to inhibit either the generation of cytotoxic effector cells by lymphoblastoid cells or lymphocyte-mediated cytotoxicity against the lymphoblasts. Although the number of HLA antigens on the surface of lymphoblasts is reported to be greater than on normal lymphocytes, the failure of alloantisera to inhibit lymphoblast-induced responses in vitro does not appear to be due to insufficient amounts of antiserum to react with the antigenic sites. Rather, the data are interpreted to suggest that antigens which are not HLA and are not closely associated with HLA on the lymphocyte membrane are responsible for the stimulation of allogeneic lymphocytes by lymphoblastoid cells. Although lymphoid cell lines are known to contain the genome of the Epstein-Barr virus, antisera against products of the viral genome fail to inhibit proliferative responses to lymphoblastoid cells, suggesting that these antigens do not directly participate in lymphocyte activation.     

Production of monospecific goat antisera to sheep IgG1 and IgG2 immunoglobulins.

Monospecific antisera to sheep IgG1 and IgG2 were produced in goats. The production of such antisera, essential for single radial diffusion technique, may be explained by the common phylogenetic origin of the two species.