Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

Effects of N-glycan processing inhibitors on signaling events and induction of apoptosis in galectin-1-stimulated Jurkat T lymphocytes

To elucidate the role of N-linked glycans in triggering T-cell functions, the effects of the N-glycan processing inhibitors 1-deoxymannojirimycin (1-DMM) and swainsonine (SW) were investigated on signaling events and induction of apoptosis in galectin-1 (gal-1)-stimulated Jurkat T lymphocytes. The treatment of Jurkat E6.1 cells with 1-DMM and SW strongly reduced the cell binding of gal-1-biotin, conjugate binding to cell lysate glycoproteins, and to cluster of differentiation (CD) 3 immunoprecipitates on blots as well as the binding of CD2 and CD3 to immobilized gal-1. The mannosidase inhibitors efficiently decreased gal-1-induced calcium mobilization. Both phases originated from a transient Ca(2+) release of internal stores, and the sustained influx across the plasma membrane was found to be involved. Both inhibitors suppressed in transiently transfected Jurkat T lymphocytes the gal-1-induced expression of the luciferase (luc) reporter gene constructs pNFAT-TA-Luc and pAP1(phorbol-12-myristate-13-acetate [PMA])-TA-Luc. The data provide evidence that gal-1 triggers through binding to N-linked glycans a Ca(2+)-sensitive apoptotic pathway.     

Inhibition of N-linked oligosaccharide processing does not prevent the secretion of thyroglobulin. A study with swainsonine and deoxynojirimycin

The effects of two drugs, swainsonine (SW) and deoxynojirimycin (dNM), on synthesis and export of thyroglobulin were studied in folliculized porcine thyroid cells cultured in a serum-free medium. These drugs were expected to alter N-linked glycans in thyroglobulin. Newly synthesized thyroglobulin labeled with [2-3H]mannose or [4,5-3H]leucine was obtained by immunoprecipitation from the follicular contents, culture media and cell extracts; the first two compartments, containing secreted thyroglobulin, were sometimes analyzed together. Leucine incorporation was not inhibited by SW and only slightly by dNM. In contrast dNM strongly decreased mannose incorporation (by up to 50-75% at 1-3 mM). However after 16-h mannose labelings, SW and/or dNM at 2.5 microM and 3 mM respectively did not significantly modify the relative proportions of radioactive thyroglobulin in the above-mentioned compartments. Pronase glycopeptides prepared from these thyroglobulins were examined with respect to behaviour on concanavalin-A-Sepharose and position on Bio-Gel P-4. Oligosaccharides released by endoglucosaminidase H and with high affinity for the lectin, i.e. high-mannose and certain hybrids, were further characterized by various exoglycosidase treatments. Thyroglobulin from control cells displayed complex and high-mannose glycans comparable in size and proportion to those attributed to tissue-extracted porcine thyroglobulin. After treatment with SW (an inhibitor of alpha-mannosidase II), complex glycans were almost totally replaced by sialylated hybrid glycans. In contrast to this nearly total suppression, dNM (an inhibitor of the trimming glucosidases) caused only a 30% decrease in labeling of complex units and an about 50% increase in high-mannose glycans, covered to some degree by glucose. Finally a [3H]leucine pulse-chase study was performed on thyroglobulin secretion in the absence or presence of both SW and dNM. Though a slowdown was detectable in the first few hours, this study revealed no change in the long-term export of thyroglobulin.     

Stimulation of Immunoglobulin Production from Human B Lymphocytes by Staphylococcus aureus: Effects of Monocytes and Con A-Induced Suppressor Cells

Significant immunoglobulin (Ig) production by human peripheral blood lymphocytes was induced in vitro by stimulating the cells with pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SpA CoI). IgG, IgM, and IgA were determined by a combination of the latex fixation test and radioimmunoassay. High levels (1,000 to 5,000 μg/ml) of IgG and IgM and a lesser amount of IgA were constantly produced during 7 to 8 days of incubation with both stimulants. Ig production induced by SpA Col stimulation was independent of the presence of T cells, while Ig production induced by PWM required T cells exclusively. Depletion of monocytes in the culture caused but a slight decrease in Ig production (particularly in the case of IgG). While the addition of a small number of monocytes enhanced IgG induction by both stimulants, coculture with an excess number of monocytes inhibited Ig induction (particularly IgG) by PWM stimulation but not by SpA CoI stimulation. Marked suppression of Ig production (IgG, IgM, and IgA) was observed in cocultures with Con A-activated T cells. The phenomena of suppression were observed in both the SpA Col-stimulated and PWM-stimulated lymphocytes. These data indicate that Ig production from B cells stimulated with a polyclonal B cell activator, SpA CoI, was independent of T cells and relatively of independent of monocytes, but could be subjected to the regulation of the Con A-induced suppressor T cells.     

Stimulation of immunoglobulin biosynthesis in human B cells by wheat germ agglutinin. I. Evidence that WGA can produce both a positive and negative signal for activation of human lymphocytes

Wheat germ agglutinin (WGA), previously regarded strictly as a nonmitogenic or anti-mitogenic lectin, can under appropriate conditions markedly stimulate in vitro synthesis and secretion of immunoglobulin (Ig) by human B lymphocytes. Stimulation of Ig production by WGA is 1) confined to a narrow lectin dose range (2 to 10 micrograms/ml; 2) abrogated by the simple sugar N-acetyl-D-glucosamine but not by a variety of other monosaccharides; 3) effective only after early additions of WGA within the initial 72 hr of 12-day cultures; 4) detected in the presence of B and T cells but not B cells alone; and 5) polyisotypic in nature, as indicated by augmented synthetic rates of Ig in each of 3 major classes (IgG, IgA, and IgM). With few exceptions, WGA produces equivalent or greater rates of Ig production as obtained in cultures activated with pokeweed mitogen (PWM), a well-recognized T-dependent polyclonal activator of human B cells. Furthermore, periperal blood lymphocytes from select individuals that respond weakly to PWM are markedly stimulated with WGA. In contrast to these stimulatory effects of WGA on Ig production by lymphocytes exposed to low lectin concentrations, addition of WGA in amounts greater than 15 micrograms/ml to PWM-stimulated human lymphocyte cultures produces marked suppression of the expected level of Ig synthesis. These data indicate that varying doses of WGA can produce contrasting stimulatory and inhibitory effects on human B cell metabolism.     

Intestinal aminooligopeptidase in diabetic BioBreed rat: altered posttranslational processing and trafficking

The structure of aminooligopeptidase (AOP), an intestinal brush-border digestive hydrolase, is abnormal in human diabetes and in the congenitally diabetic BioBreed Wistar (BB(d)) rat. Its assembly in the BB(d) rat was examined. After normal initial synthesis and assembly of immature AOP precursor (AOP(i)) with high-mannose N-linked chains in the endoplasmic reticulum (ER), processing of N-linked glycans in Golgi yielded a smaller than normal mature AOP precursor (AOP(m)) with persistence of some high-mannose N-linked chains. Deglycosylation analyses suggested that the mass difference could be attributed to a lower mass of N-linked with unaltered O-linked glycans in AOP(m) of the diabetic rat. Intrajejunal pulse-chase experiments revealed that the conversion of AOP(i) to AOP(m) occurred at 30 min of chase in normal rats but at 60-90 min in diabetic rats, reflecting delay in ER-to-Golgi transport or a slower processing of high-mannose chains. Once maximal transport to Golgi was achieved, the residence time in Golgi was shortened in diabetes. This altered processing of the precursor accounted for the altered structure of AOP in diabetes.     

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.     

Contribution of leptin receptor N-linked glycans to leptin binding

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.     

Monoclonal antibody against the human peripheral lymph node homing receptor homologue (Leu 8) inhibits B cell differentiation but not B cell proliferation.

Previous studies have shown that a subpopulation of circulating human B cells expresses the Leu 8 peripheral lymph node homing receptor homologue and that these B cells are capable of producing Ig in response to staphylococcus A Cowan I (SAC). In the present study the effect of a signal delivered via the Leu 8 molecule (using anti-Leu 8 mAb) on B cells was examined. Initially, it was shown that immobilized anti-Leu 8 suppressed IgM and IgG secretion of B cells activated by SAC + IL-2 but not that by PWM-prestimulated B cells or B cells stimulated with PWM in the presence of CD4+, Leu 8- T cells (a source of helper cells). It was also shown that anti-Leu 8 did not suppress SAC + IL-2-stimulated B cell proliferation or expression of IL-2R alpha-chain or c-myc mRNA in B cells. The addition of T cells, monocytes, purified IL-2, rIL-1, rIL-6, or human B cell growth factor did not overcome the inhibitory effect of anti-Leu 8 on SAC-stimulated B cell Ig production, and the inhibitory effect of anti-Leu 8 was not blocked by anti-TGF-beta. Finally, inhibition of B cell differentiation occurred even when anti-Leu 8 was added up to 72 hrs after initiation of cell culture. Thus, anti-Leu 8 is unique among inhibitors of B cell function in that it can down-regulate immunoglobulin synthesis without affecting B cell proliferation. These findings suggest that a natural ligand for Leu 8 could affect not only homing of B cells, but also B cell differentiation.     

Synthesis of enzymatically active human alpha-L-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds

As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T(2) seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 microg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 microg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans.     

Swainsonine, an inhibitor of mannosidase II during glycoprotein processing, enhances concanavalin A-induced T cell proliferation and interleukin 2 receptor expression exclusively via the T cell receptor complex.

The T cell receptor (TCR) is a disulfide-linked heterodimer consisting of both complex and high-mannose types of N-linked oligosaccharides. The objective of the present investigation was to examine the effect of altered oligosaccharide structure on the expression and function of the TCR. Human mononuclear lymphocytes (MNL) were treated with castanospermine (CAST) or swainsonine (SW), inhibitors of glucosidase I or mannosidase II, respectively. Treatment with these inhibitors does not prevent glycosylation, but results in synthesis of glycoproteins with high-mannose or hybrid types of oligosaccharides. Treatment of MNL with CAST (1000-10 microM) or SW (100-1 microM) for up to 72 hr had no effect on cell surface expression of of the TCR. SW potentiated Con A-induced T cell proliferation without effecting anti-CD3 (OKT3) or alloantigen-induced proliferation. CAST had no effect on Con A, anti-CD3, or alloantigen-induced T cell proliferation. The T cell proliferative response to Con A in the presence of SW was completely eliminated in the presence of monoclonal anti-TCR antibodies. Monoclonal anti-CD2, -CD3, -CD4, -CD8, or isotypic control monoclonal antibodies had no effect on SW enhancement of T cell proliferation. SW treatment potentiated Con A-induced MNL expression of both the alpha and beta subunits of the IL 2R. This effect was also specifically blocked by anti-TCR monoclonal antibodies. These results demonstrate that selective changes in the glycosylation state of the TCR complex can alter mitogen recognition and subsequent cellular activation.     

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.     

Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.

The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.     

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.     

N-linked glycans of the human insulin receptor and their distribution over the crystal structure

The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.     

Production of human suppressor T cell hybridomas

To study human T cell suppression of immunoglobulin (Ig) synthesis with homogeneous populations of immunoregulatory cells, human suppressor T cell hybridomas were prepared by somatic cell fusion of concanavalin A-activated peripheral blood T cells with hypoxanthine-guanine phosphoribosyltransferase-(HGPRT, EC 2.4.2.8) deficient human leukemic CEM T cells. After selection in hypoxanthine-aminopterin-thymidine (HAT) medium and cloning by limiting cell dilution, two human T cell hybridomas were identified that produced 60 to 80% suppression of in vitro polyclonal immunoglobulin production when cocultured with pokeweed mitogen- (PWM) stimulated peripheral blood lymphocytes. Further, one of the suppressor T cell hybridomas constitutively secreted a soluble suppressor factor(s) (TsF) of m.w. 70,000 to 85,000 daltons, which produced reversible noncytotoxic inhibition of lectin-activated B cell Ig production. In contrast, this TsF did not inhibit lectin- or antigen-induced T cell proliferation, nor did it interfere with the generation or effector function of cytotoxic T cells. Additional studies indicated that this Tsf acts directly on B cells or monocytes rather than indirectly modulating the activity of immunoregulatory T cells. In summary, these studies suggest that techniques of somatic cell fusion may provide a valuable approach to further study human immunoregulatory cell-cell interactions as well as provide a source of sufficient quantities of important lymphokines for further purification and characterization.     

Expression of VHIII-associated cross-reactive idiotype on human B lymphocytes. Association with staphylococcal protein A binding and Staphylococcus aureus Cowan I stimulation.

It has been demonstrated that staphylococcal protein A (SPA) has an "alternative" binding site with specificity for human Ig H chain V region of the VHIII subgroup. Because the major mitogenic component of Staphylococcus aureus Cowan I (SAC) is SPA, it is possible that SAC stimulates a subpopulation of B cells expressing Ig of the VHIII H chain subgroup. In the present study, we have investigated further the relationship between SPA binding and the expression of VHI- or VHIII-associated cross-reactive idiotype (CRI) on the surface of tonsillar B lymphocytes enriched for the expression or nonexpression of the CRI, and we examined the Ig secreted by cell lines established from these populations of B cells by EBV transformation. The VHIII CRI (D12)-enriched population yielded 21 cell lines, with 67% of them secreting SPA-reactive Ig; in contrast, only 6% (1 of 16) of VHI CRI-expressing lines secreted SPA-reactive Ig. The CRI-negative B cell population yielded 54 cell lines, of which 20% secreted SPA-reactive Ig, as might be anticipated because a majority of VHIII Ig+ B cells will be CRI-. SAC stimulation of CRI+ and CRI- populations showed preferential stimulation of the D12 population. These data support the proposal that SAC stimulation of human B cells is mediated through binding of SPA by its alternative binding site to IgV regions of the VHIII subgroup.     

Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.