Binding of bacterial lipopolysaccharide to histocompatibility-2-complex proteins of mouse lymphocytes.

The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells.     

Interferon inducing transformed cell surface glycoproteins: purification by Ia antigen affinity chromatography

Ia antigens were previously shown to be B-cell receptors for transformed cell surface glycoproteins (TCSG). Ia antigen recognition of TCSG subsequently initiated interferon production. These findings have been exploited to highly purify TCSG from mouse L (H-2k) cells in one step by Iak antigen Sepharose affinity chromatography. These results point to the highly specific recognition of TCSG by Ia antigens and the B-cells on which they reside. The implications and applications of these studies are 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.     

Defective T-cell activation by Mycoplasma arthritidis mitogen is restored by interferon-gamma

A mitogen derived from supernatants of Mycoplasma arthritidis (MAS) has been shown to induce both T-cell activation and the production of interferon-gamma (IFN-gamma). MAS-induced response required the presence of accessory cells and is under the genetic control associated with the major histocompatibility complex (MHC). We found that recombinant IFN-gamma restored the proliferative response to MAS mitogen in unreactive mice strains, including H-2b and H-2s haplotypes. We postulated that these T-cells fail to respond since they lack part of the I-E molecules on their accessory cells. Our data suggest that interferon-gamma may be able not only to increase the levels of Ia antigens but also to promote the appearance of MHC products that are not usually present on the cell surface. Since Ia antigens have a central role on T-cell activation, we examined the effect of the level of IFN-gamma on MAS-induced T-cell activation. We analyzed the acquisition of responsiveness to IL-2 and IL-2 activity in cells pretreated with IFN-gamma and found that both the steps of T-cell activation were restored to the MAS mitogen in the unreactive mice strains by IFN-gamma.     

Carbohydrate-binding specificity of pokeweed mitogens

The carbohydrate-binding specificity of two pokeweed (Phytolacca americana) mitogens (Pa-1 and Pa-2) was investigated by means of hemagglutination inhibition assays and the quantitative inhibition of the binding of 125I-labeled lectins to human erythrocytes using various oligosaccharides, glycopeptides and glycoproteins as hapten inhibitors. Among the inhibitors employed in this study, chitin oligosaccharides and the glycopeptides and glycoproteins which bear sugar chains of the type found in serum glycoproteins, particularly PAS-1 glycoprotein and band-3 glycoprotein of human erythrocyte membranes, exerted strong inhibitory activity. The inhibitory constants of band-3 glycoprotein toward the binding of both mitogens to human erythrocytes were found to be very close to the association constants of the mitogens to the cells. Furthermore, the results of competitive binding studies between Pa-1 and Pa-2 indicated that these mitogens share a common oligosaccharide chains on the erythrocyte surface. To isolate the membrane receptors for these two mitogens, the solubilized membranes of human erythrocytes were subjected to affinity chromatographies using Pa-1-Sepharose 4B and Pa-2-Sepharose 4B as specific adsorbents. In both cases of these two specific adsorbents, band-3 glycoprotein was found to bind most strongly. These results suggest that two pokeweed mitogens have essentially the same carbohydrate-binding specificity and they bind primarily to the sugar chains of band-3 glycoprotein, possibly to the core structure of the sugar chains containing a di-N-acetylchitobiose moiety, on human erythrocytes.     

Susceptibility to lipid peroxidation and accumulation of fluorescent products with age is greater in T-cells than B-cells

The age-related loss of immune function, which is primarily due to loss of T-lymphocyte function, is also associated with accumulation in spleen lymphocytes of autofluorescent products indicative of peroxidation damage. In this study, we examined T-cell membranes for age-related changes which could be related to lipid peroxidation. Using fluorescence spectroscopy of CHCl3:CH3OH membrane extracts, we observed that old T-cells have a 2-fold greater accumulation of fluorescent products than old B-cells and that young T-cells, when exposed to free radicals in an in vitro system, accumulate significantly more fluorescent products over time than young B-cells. We used fluorescence polarization to show that young T-cell membranes are more fluid than young B-cell membranes. However, T-cell membrane fluidity decreases with age, whereas B-cell membrane fluidity does not change; in old mice, T-cell membranes are significantly less fluid than old B-cell membranes. Using two-dimensional electrophoresis, we showed that membrane extracts of old T-cells contain many more proteins than extracts of young T-cells. Our results indicate that age-related changes occur in T-cell membranes which could be due to their increased susceptibility to lipid peroxidation and these changes may contribute to the age-related decline in immune function.     

The lectin binding sites on the plasma membrane components of human lymphoblastoid cell lines.

The plasma membrane components of five human B-cell lines and three human T-cell lines were separated by dodecyl sulfate polyacrylamide gel electrophoresis, incubated with the radioactive labeled lectins from lentil, castor bean, wheat germ, Phaseolus bean, peanut, gorse and the Roman snail and the molecular weights of the binding sites determined. The lentil, castor bean and wheat germ lectin bound to multiple components from molecular weights (Mr) 20 000 to 200 000 within the plasma membranes, whereas peanut lectin bound preferentially to glycoproteins of Mr 150 000 and 83 000 in B-cells, and 150 000 and 130 000 in T-cells. The gorse lectin bound to a 220 000 component in B-cells which was not labeled in T-cells.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.     

Surfaces of murine lymphocyte subsets differ in sialylation states and antigen distribution of a major N-linked penultimate saccharide structure

Rat liver beta-galactoside alpha-2,6-sialyltransferase and Vibrio cholerae sialidase were used with cytidine-5'-monophospho-N-acetyl-[3H]neuraminic acid (CMP-[3H]NeuAc) to specifically probe the distribution and sialylation state of Gal beta 1-4GlcNAc residues on N-linked saccharides on the surfaces of murine lymphocytes. The relative extent of exogenous sialyltransferase-mediated sialylation (per cellular protein) was thymocytes greater than T-cells greater than T-cell lymphoma (EL-4) greater than B-cells greater than B-cell lymphoma (AKTB-1b) greater than splenocytes. Prior desialylation increased exogenous resialylation by 23.8-, 13.1-, 7.1-, 7.9-, 7.0-, and 5.3-fold for splenocytes, B-cells, T-cells, EL-4, AKTB-1b, and thymocytes, respectively. Though numerous glycoproteins were labeled, the majority of the Gal beta 1-4GlcNAc residues were detected on a relatively small number of cell surface proteins, many of which are well-defined lymphocyte antigens. Gal beta 1-4GlcNAc residues on thymocytes were found to exist in an undersialylated state on T200 but not on other antigens (e.g., Thy-1). T200 was found to be fully sialylated on mature cells (i.e., hydrocortisone-resistant thymocytes and splenic T-cells), suggesting that its sialylation state is developmentally regulated. These studies indicate that the number, sialylation state, and polypeptide distribution of the penultimate structure, Gal beta 1-4GlcNAc, differ on N-linked saccharides on the surfaces of different lymphocyte populations.     

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.     

Mitogens and T-independent antigens stimulate T lymphocytes to secrete La antigens.

Previous reports from this laboratory suggest that certain I region-associated (Ia) antigens can be detected in normal mouse serum. It was found that, when mitogens are injected into mice, they produce substantial increases (up to 125-fold) in the levels of these Ia antigens in mouse serum. Similar increases were obtained when either T- or B-cell mitogens were injected. Furthermore, in vitro and in vivo studies demonstrated that the mitogens stimulated T cells to secrete Ia antigens. It appears likely, however, that the Ia antigens detected in these studies may differ from the conventional Ia glycoproteins found on the surface of B lymphocytes.All T-independent antigens tested also augmented the concentrations of Ia antigen in serum, the increases depending on the T-independent antigen injected and ranging from 3- to 125-fold. In contrast, T-dependent antigens, unless injected in large amounts, were unable to produce detectable changes in the serum levels of Ia antigen. These data indicate that an inverse relationship exists between the T dependence of an antigen and its ability to stimulate T cells to secrete Ia antigens. On the basis of this conclusion it is proposed that all antigens are T dependent and merely vary in the efficiency with which they activate T cells to release helper factors.     

The role of human interleukin-6 in B-cell isotype regulation and differentiation

Human Interleukin-6 (IL-6) is a cytokine secreted by T cells, as well as a variety of other cell types, which exhibits B-cell differentiating activity. The recent cloning of the gene that codes for this molecule has allowed us the opportunity to study the function of this molecule alone and in conjunction with other lymphokines in human B-cell isotype-regulation and differentiation. Recombinant human IL-6 enhances immunoglobulin (Ig) M and G secretion by B-cells activated by Staphylococcal A Cowan strain (SAC) and enhances IgM, IgG, and IgA secretion by B-cells activated by pokeweed mitogen. IL-6 also augments immunoglobulin secretion of differing isotypes from various Epstein-Barr Virus transformed B-cell lines. However, IL-6 does not alter the secreted isotype of naive surface IgM-positive B-cells. As human T-cells secrete other lymphokines in association with IL-6 after activation we examined the interaction of Interleukin-2 (IL-2) gamma-interferon (IFN-gamma) and Interleukin-4 (IL-4) with IL-6 on B-cell immunoglobulin secretion. IL-2 and IL-4 synergized with IL-6 in augmenting immunoglobulin secretion by SAC-activated B-cells. IFN-gamma significantly inhibited the Ig secretion of SAC-activated B-cells cocultured with IL-6 alone or in combination with IL-2. These results demonstrate that human recombinant IL-6 augments immunoglobulin secretion of isotype-committed B-cells but it does not induce a change in the isotype secreted. In addition, this lymphokine synergizes with IL-2 and IL-4 in supporting Ig secretion. However, IFN-gamma significantly inhibits IL-6 induced Ig secretion.     

Human helper-T-cell function does not require T4 antigen expression

The relationship between immunoregulatory T-cell function and the expression of T-cell subset-specific differentiation antigens was examined using a phenotypically anomalous human T-cell line (TCL), termed H-1. H-1 cells were found to express T11, extremely high levels of T3, but no T4 nor T8 antigen. Despite their lack of T4 antigen expression, H-1 cells could be activated by coculture with pokeweed mitogen (PWM), anti-T3 antibody, or autologous B cells to provide potent help for B-cell differentiation into plaque-forming cells (PFC). In contrast, H-1 cells did not suppress the PFC response triggered by PWM-activated T4+ cells. These results demonstrate that the expression of the T-cell subclass-specific differentiation antigen, T4, is not required for a T cell to become activated and to implement the program for helper function. In addition, enhanced expression of T3 on the T4-, T8-, H-1 cell surface may reflect a compensatory upregulation of the T3/Ti receptor complex on T cells which are deficient in these nonpolymorphic associative recognition structures.     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

T-cell hybridoma secreting hemopoietic regulatory molecules: granulocyte-macrophage and eosinophil colony-stimulating factors.

Murine spleen cells stimulated in vitro with pokeweed mitogen were fused with a HAT-sensitive AKR thymoma (BW5147) to produce T-cell hybridomas secreting hemopoietic colony-stimulating factors (CSFs). A stable cloned T-cell hybridoma has been isolated which expressed the H-2 antigens of both fusion parents, has a median chromosome number of 56 and secretes a factor(s) which stimulates the growth of granulocyte-macrophage and eosinophil colonies. The CSF-secreting hybridoma exhibited only the Thy 1.1 associated with the parent tumor, but no markers normally associated with normal T-cells or macrophages were detected. No CSF was secreted by the parent tumor line, but the hybridoma-conditioned medium, when used at 10% (v/v), contained sufficient CSF to stimulate 10–30 colonies per 10⁵ bone marrow cells. Lipopolysaccharide (1 μg/ml) stimulated the production of CSF by the hybridoma cells 3 fold. CSF production also increased when the cells were held at high density in serum-free medium. The colony-stimulating factor(s) secreted by the hybridoma exhibited similar molecular properties to those produced by pokeweed mitogen-stimulated spleen cells, and both the GM- and EO-CSFs had an apparent molecular weight by gel filtration of approximately 35,000.     

Demonstration of Ia antigens on mouse intestinal epithelial cells by immunoferritin labeling

Several kinds of epithelial cells that express H-2 antigens were studied by immunoferritin labeling with an antiserum reacting only with antigens of theI region of theH-2 complex. Spleen lymphocytes were used to test the labeling system and the effect of the epithelial cell dissociation procedure on Ia antigens. Immunoglobulin-positive B10.BR lymphocytes were labeled with an anti-la^k serum (A.TH anti-A.TL serum absorbed with BALB/c and B10.D2 cells), while congenic B10.D2 lymphocytes were unlabeled. The distribution of labeled Ia antigens on living B10.BR lymphocytes was patchy, while on cells fixed in periodate-lysine-paraformaldehyde before labeling, the distribution of label was continuous. Fixation evidently immobilized Ia antigens in the lymphocyte membrane. Trypsin and collagenase, as used in the epithelial cell dissociation procedure, had no discernible effect on the Ia antigens of lymphocytes. The epithelial cells studied included the columnar absorptive cells of the small intestine, uterine lining epithelium, tracheal brush cells, and pancreatic exocrine and duct cells. These cells were fixed before dissociation from their respective tissues. Ia antigens were detected only on the columnar absorptive cells of the small intestine. These cells labeled equally well with an antiserum reacting only with theK -end of theH-2 complex. In both cases, congenic control intestinal cells were unlabeled. Thus, intestinal epithelial cells appear to express theIa, K, and presumablyD regions of theH-2 complex, while the other epithelial cell types express only the K and D antigens. On fixed intestinal epithelial cells, Ia and H-2K antigens were continuously distributed on the lateral and basal cell membranes including the zonula adherens, but the antigens were absent from the apical microvillous membrane and the zonula occludens.     

Selective activation of human B-lymphocytes by suboptimal doses of pokeweed mitogen (PWM). Quantitation and ultrastructure of the stimulated cells

The activation and differentiation of human peripheral blood lymphocytes, by various doses of pokeweed mitogen (PWM) were studied in vitro by thymidine uptake and electron microscopy. Quantitative and morphological observations on B-cell depleted and unfractionated lymphocyte populations indicated that B-cells were rapidly activated and differentiated to plasmablasts and plasmacytes by suboptimal doses of PWM. In contrast, B-depleted lymphocyte cultures showed a significant delay in thymidine incorporation and transformation rate. At high doses of PWM both B-cell depleted and unfractionated lymphocyte cultures had approximately the same levels and kinetics of thymidine incorporation and transformation rate. Differentiation to plasmacytes was not observed in B-depleted lymphocyte preparations.     

Characteristics of T-cells synthesizing macrophage migration inhibition factor in an H-2 system]

Migration inhibition factor (MIF) was found to be produced by T-cells rather than B-cells as shown by the indirect macrophage migration inhibition test in the H-2 system performed with fractionated lymphocytes. MIF-producers appeared to be more sensitive than T-killer cells to the cytotoxic action of anti-theta-antibodies, plus complement. MIF synthesis by lymph node cells developed earlier after immunization than the cytotoxic activity of these cells. These findings indicated that T-cell subpopulation producing MIF differed from the cytotoxic T-lymphocytes.     

The cellular basis for viral-induced immunodeficiency: analysis by monoclonal antibodies

Viral infections are often associated with immunodeficiency states. Although T lymphocytes have been thought to suppress the host's response, the precise etiology remains unclear. Therefore, we characterized T lymphocytes from six patients during both acute and convalescent phases of infectious mononucleosis (IM) with monoclonal antibodies (titer, 10(-5) to 10(-7) to antigens restricted to the TH2- helper (T4) and TH2 suppressor (T5) T cell subsets as well as to a common T cell antigen (T3) and HLA-D related Ia antigens. It was found that during acute infectious mononucleosis, there is both activation and increase of suppressor T cells (T5+, Ia+ phenotype). Fuctionally, the acute IM lymphocytes suppress autologous T cell proliferation to antigens as well as pokeweed mitogen driven B cell immunoglobulin production. In contrast, convalescence is associated with a return to normal of T cell subsets and immune function. These results demonstrate that viral infections can preferentially activate a specific T cell subset and suppress the overall human immune response.