Antigen Binding Lymphocytes in Congenitally Athymic (Nude) Mice

THE autoradiographic detection of the binding of various radiolabelled antigens to a proportion of lymphocytes from animals not exposed to those antigens (“nonprimed” lymphocytes) is well documented^1–4. Such lymphocytes are thought to have patches of surface immunoglobulin, primarily IgM, which act as specific receptors for antigen^5,6. A proportion at least of these unprimed lymphocytes are immunologically competent as shown in vivo^7,8 and hence are true antigen reactive cells. Most assays have used peripheral lymphocyte suspensions from tissues of man, mouse, rat and chicken, not enriched or fractionated in any way for the two distinct lines of lymphocytes, thymic derived (T) and non-thymic derived (B)⁹. It is not clear whether antigen-binding cells (ABC), detected in routine assays where autoradiographs are exposed for 1–2 weeks, are of both T and B cell type or are predominantly of only one type. Experiments using unlabelled and radiolabelled immunoglobulin antisera with isolated T and B cells have inferred specific antigen binding on both populations although T cells seem to have far fewer antigen binding receptors than B cells¹⁰.     

Specific Alteration of the Surface Charge of Immunocytes

THERE is increasing experimental evidence that the receptor groups of immunocytes which react with a specific antigen are analogous to antibody molecules capable of binding the antigen¹. This property has been used to advantage in attempts to isolate lymphocytes possessing a distinct immunological potential by their adsorption to and subsequent elution from appropriate immunosorbents².     

Reversible Blocking Effect of Anti-mouse Immunoglobulin Serum on the Induction of Immunity and Tolerance <Emphasis Type="Italic">in vitro</Emphasis>

THE induction of blast transformation by incubating lymphocytes with anti-immunoglobulin¹ and anti-allotype² sera has suggested that these cells have immunoglobulin on their surface. This hypothesis was directly verified by the demonstration of immunoglobulin on living mouse lymphoid cells by Raff et al.³. There is much evidence to indicate that immunocompetent cells have surface receptors for antigen. This idea is based on the finding that lymphocytes can bind radioactively labelled antigen to their surface^4,5 and that specific immune unresponsiveness occurs if lymphoid cells are exposed to either highly radioactive antigen⁶ or haptens capable of forming covalent bonds with proteins^7,8. The immunoglobulin nature of these antigen receptors is suggested by recent work showing that the binding of radioactively labelled antigen can be blocked by anti-immunoglobulin sera^5,9. Reports that the adoptive immune response of mouse spleen cells can be inhibited by anti-mouse immunoglobulin sera (AMS)^9,10 suggest that the interaction of antigen with the immunoglobulin receptor sites is a crucial step in the induction of the antibody response. We report here that the inhibitory action of AMS on the immune response is potentially reversible and that the induction of immune tolerance to polymerized flagellin (POL) in vitro may be blocked in the presence of AMS.     

Isolation of Surface Immunoglobulin from Lymphocytes from Human and Murine Thymus

CELLS capable of binding radioiodinated antigen occur in lymphocyte populations obtained from foetal and neonatal human thymus¹. In accordance with Ehrlich's hypothesis of 1900 (ref. 2), the receptors for antigen on the surface of these cells probably consist of immunoglobulin. A first step in the attainment of chemical proof of the nature of this receptor is the identification of immunoglobulin on the thymus cells. We now report the isolation of immunoglobulin from the surfaces of lymphocytes obtained from murine and neonatal human thymuses. This isolation and identification was made possible by application of a modification³ of the enzymatic iodination procedure of Marchalonis⁴ which catalysed the iodination of proteins present on the surfaces of living lymphocytes.     

Surface-bound Immunoglobulin as a Marker of B Lymphocytes in Man

IMMUNOLOGICAL reactions in many species depend on two functionally different types of small lymphocytes, thymus-derived T lymphocytes and bone marrow-derived B lymphocytes¹. In the mouse, the θ antigen is a marker for T lymphocytes, while membrane-bound Ig is found on θ-negative lymphocytes². This has raised the question whether the presence of surface-bound Ig³ may be regarded as a marker for B lymphocytes. We have obtained data indicating that this is the case with human lymphocytes.     

Inability of EDTA to prevent damage mediated by cytolytic T-lymphocytes.

To analyze the nature of the target cell determinants recognized and bound by killer lymphocytes during lymphocyte-mediated cytolysis (LMC), the specific binding of serologically active tumor cell membrane fractions to cytotoxic T lymphocytes has been investigated. Particulate membrane fractions and soluble antigen preparations (extracted by papain or 3 M KCl) from tumor target cells were tested for their ability to inhibit the destruction of intact ⁵¹Cr-labeled target cells by killer lymphocytes in vitro. The effect of papain-solubilized tumor cell antigen on the binding of killer lymphocytes to tumor cell monolayers was also evaluated. Direct assays to determine the extent of binding of unlabeled or radioiodinated soluble antigen (extracted by papain or deoxycholate) to cytotoxic lymphocytes were carried out. In marked contrast to their serological activity, all of these particulate and soluble preparations failed to inhibit LMC or bind to killer lymphocytes in an immunologically specific way. It is suggested that killer lymphocytes recognize and bind to an antigenic complex whose organization is dependent upon the integrity of the target cell membrane.     

Mechanism of binding of soluble immune complexes to lymphocytes

Soluble immune complexes prepared with either (a) ¹²⁵I BSA and mouse antiserum to BSA in the presence of fresh mouse serum (AgAbC) or with (b) ¹²⁵I BSA and a 7S fraction of the mouse antiserum to BSA (AgAb) bind to a certain proportion of mouse lymph node and spleen lymphocytes (but not to thymocytes). The efficiency of binding is greater when complexes are prepared at defined antigen/antibody ratios and when the incubation with lymphocytes is performed at 37 °C. However, a significant degree of binding occurs at lower temperatures and even at 0 °C. Cells which bind soluble complexes overlap extensively with complement receptor lymphocytes (CRL) (B lymphocytes) since the specific elimination of CRL also depletes the population of cells which can bind soluble complexes. Two types of interactions are involved in the binding: one mediated by antibody which has been aggregated by antigen and another by complement (probably C3). They can be operationally distinguished because (1) after treatment of the lymphocytes with trypsin, the binding of AgAbC (but not of AgAb) is strongly inhibited; (2) the binding of AgAb (but not of AgAbC) is inhibited by heat-aggregated Ig; (3) the binding of AgAbC (but not of AgAb) to lymphocytes inhibits their subsequent interaction and rosette formation with erythrocytes sensitized by antibody and complement components; and (4) cobra venom factor markedly alters the binding of AgAbC to lymphocytes.     

Properties of Educated T Cells for Rosette Formation and Cooperation with T Cells

IT has been shown that the humoral antibody response in mice to many antigens requires cooperation between thymus derived lymphocytes (T cells) and bone marrow derived lymphocytes (B cells)^1,2. The B cells are the direct precursors of antibody secreting cells and, although T cells react specifically with antigen, their role is unknown^2–6.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Root Cap and Georeaction

CIRCULATING lymphoid cells suffer alterations in a localized disease, chronic viral keratitis; we have established long term lymphoctye cultures from the blood of such patients¹. We have used capillary migration technique to study cellular immunity in the disease. Leucocytes from patients were challenged with corneal antigen and with the virus which had initiated the infection^2,3. Migration inhibition is specific for the antigen to which the animal is sensitive and on contact with antigen, sensitized lymphocytes produce migratory inhibitory factor (MIF).     

Induction of Long Term Lymphocyte Lines from Delayed Hypersensitive Human Donors using Specific Antigen plus Epstein–Barr Virus

THE availability of homogeneous populations of human and murine myeloma cells has provided a unique opportunity for investigating the mechanism of immunoglobulin formation¹. Continuous lines of cultured lymphoid cells producing specific antibody or manifesting delayed hypersensitivity would be even more useful in studying the molecular events of the immune response. Human lymphoid cell lines have been established in long term culture using Epstein–Barr virus (EBV)^{2, 3} or phyto-haemagglutinin⁴ but antigen alone has not been effective⁵. The purpose of the work reported here was selectively to establish antigen-sensitive cells in culture by stimulating peripheral white cells from delayed hypersensitive donors with antigen in vitro and then exposing the cells to EBV. This combination of antigen and virus was chosen because of the following considerations: (1) some RNA and DNA viruses do not replicate in resting lymphocytes but can infect antigen-sensitive lymphocytes which have been stimulated in vitro with mitogens or specific antigen^{6, 7}; (2) polyoma virus transforms cells in the G₂ phase of the cell cycle more effectively than in G₁ (ref. 8). These observations suggested that combined exposure to antigen and EBV might result in the establishment of cell lines enriched for antigen-sensitive or antibody-forming cells.     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.     

Specific Collaboration between T and B Lymphocytes across a Cell Impermeable Membrane <Emphasis Type="Italic">in vitro</Emphasis>

ANTIBODY production to many antigens including heterologous erythrocytes^1–3, serum proteins^4,5 and hapten protein conjugates^6,7, occurs as a result of an interaction between antigen and thymus-derived (T) and non-thymus-derived (B) lymphocytes. Although the specificity of the antibody response is determined by T as well as B cells^8–10, T cells do not actively secrete any of the known classes of immunoglobulin molecules¹¹. Their function seems rather to initiate the sequence of events whereby antigen is presented to B cells in an immunogenic form capable of stimulating antibody synthesis¹².     

Beta-adrenoceptors of human lymphocytes: decrease in affinity of agonist for (-)3h-dihydroalprenolol binding sites by cytoplasmic fractions

The beta-adrenoceptors of intact human lymphocytes were investigated by binding assays with a radiolabeled β-adrenergic antagonists, (?)³H-dihydroalprenolol (DHA). Results shown are 1.48 ± 0.57 × 10⁴ binding sites for the ligand per cell. Binding of (?)³H-DHA to the membrane fractions of human lymphocytes was studied for comparison. The affinity of (?)³H-DHA for membrane was similar to that for whole lymphocytes, but the binding to the membranes was inhibited more strongly by β-adrenergic agonist. Moreover, the cytoplasmic fraction of human lymphocytes decreased the inhibition by (?)-isoproterenol of (?)³H-DHA binding to the membrane fraction. The results imply that the cytoplasmic fraction of human lymphocytes lowered the affinity of (?)-isoproterenol to (?)³H-DHA binding sites and left no effect on the binding of the β-adrenergic antagonist to the sites.     

Concanavalin A as an Inducer of Human Lymphocyte Mitogenic Factor

IT is likely that pharmacological products of antigen : lymphocyte interaction (“lymphokines”) act as mediators and regulators of a variety of cellular immune responses^1,2. This view is strengthened by demonstrations that phytomitogen lectins induce lymphocytes to generate products with similar biological activities^3,4 and physicochemical characteristics⁵, to the lymphokines. Increasing evidence suggests that mitogenic lymphokines may mediate lymphocyte transformation responses in vitro and facilitate lymphoid cell cooperation in vivo (refs. 1,2,6–9). The study of mitogenic factor production by phytomitogens which may predominantly activate thymus-dependent lymphocytes (Concanavalin A (ConA))^8,9 provides a model approach to the investigation of lymphokine function in man. Powles et al.⁴ have described a ConA-induced mitogenic factor which stimulated autologous human lymphocytes only, whereas antigen-induced lymphocyte factors generally stimulate both allogeneic and syngeneic lymphocytes^11–13. Interest in ConA as an inducer of human lymphocyte mitogenic factors would be widened if conditions were found in which ConA stimulated human lymphocytes to generate products which were mitogenic for both allogeneic and autologous lymphocytes. As a lymphokine stimulant, ConA has the advantage that it is largely removed from culture fluids by absorption to cross-linked dextrans (‘Sephadex G-50’) or serum glycoproteins¹⁴. Here we demonstrate that a ‘Sephadex’-binding fraction of ConA (ConA- V) induces human lymphocytes to generate a mitogenic factor which activates both allogeneic and autologous lymphocytes.     

Effects of mitogens on sodium-potassium transport, H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na⁺) potassium (K⁺) transport, ³H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K⁺ pump influx, while the cardiac glycoside-insensitive K⁺ leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K⁺ pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na⁺ efflux in human lymphocytes. Apparently these mitogens activate the Na⁺K⁺ pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na⁺K⁺-ATPase. From rate studies of ³H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na⁺K⁺ pumps.     

N-Acetylcysteine inactivates Migration Inhibitory Factor and Delayed Hypersensitivity Reactions

In vitro studies suggest that delayed hypersensitivity follows the production of migration inhibitory factor (MIF) by sensitive lymphocytes in the presence of specific antigen. This factor arrests the migration of macrophages in vitro and in vivo. After attraction, aggregation and activation in vivo, these bystander cells produce toxic substances which induce the local reaction¹. When lymphocytes from tuberculin (PPD) sensitized guinea-pigs were incubated with PPD, cell-free supernatant fluids of the cultures contained MIF². Such migration inhibitory fluids injected intradermally with PPD, into PPD-sensitive animals, enhanced the delayed hypersensitivity reaction³. Concentrated migration inhibitory supernatant fluids injected intradermally into unsensitized animals produced local reactions of induration and erythema within 6 h; reactions reached a maximum after 16 h. Histologically there was an infiltrate of mononuclear cells at the site of injection and neutrophils and eosinophils were also present¹.     

Thymus and Bursa Dependence of Lymphocyte Mitogenic Factor in the Chicken

AMONG the soluble products generated during activation of rodent or human lymphocytes¹, lymphocyte-stimulating (‘mitogenic’) factors are detected by their ability to accelerate DNA metabolism of freshly cultured allogeneic or syngeneic lymphocytes^2–8. If such lymphocyte activation products function as mediators of cellular immunity^1,9–11, their activities will be generated independently of the antibody-forming system. In the chicken, antigen-stimulation of sensitized lymphocytes in vitro involves both thymus (T-) and bursa-(B-)-dependent cells^12–14. If antigen-induced lymphocyte transformations are generally mediated by lymphocyte mitogenic factors, both T-cells and B-cells should produce lymphocyte mitogenic factors when stimulated by specific antigen. We have therefore determined whether antigen-stimulated chicken lymphocytes generate a lymphocyte mitogenic factor and whether this response is affected by neonatal thymectomy or bursectomy.