Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells

Regulatory T cells (Tregs) can potentially migrate to the B cell areas of secondary lymphoid tissues and suppress T cell-dependent B cell Ig response. T cell-dependent Ig response requires B cell stimulation by Th cells. It has been unknown whether Tregs can directly suppress B cells or whether they must suppress Th cells to suppress B cell response. We report here that Foxp3+ Tregs are found in T-B area borders and within germinal centers of human lymphoid tissues and can directly suppress B cell Ig response. Although Tregs can effectively suppress T cells, they can also directly suppress B cell response without the need to first suppress Th cells. The direct suppression of B cell Ig production by Tregs is accompanied by inhibition of Ig class switch recombination.     

Capsaicin does not attenuate bombesin-induced suppression of operant responding for food reward.

Systemic treatment with capsaicin, a neurotoxin which damages unmyelinated peptide-containing sensory neurons, has been shown to attenuate bombesin (BBS)-induced suppression of food intake. To determine whether capsaicin-sensitive fibers mediate the effect of BBS on appetitive motivation, we examined BBS-induced suppression of operant responding in rats pretreated neonatally with capsaicin (50 mg/kg; SC) or control vehicle. At 8-10 weeks of age, rats were trained to bar press for food. After achieving a stable level of performance, the animals were injected with BBS (10 micrograms/kg), normal saline, or prefed with 20 Noyes 45-mg pellets. Animals were then tested in an operant chamber on an FR 5 schedule of reinforcement for one hour. The results indicated that BBS suppressed bar pressing, regardless of whether animals were pretreated with capsaicin or control vehicle. These findings are inconsistent with the hypothesis that BBS induces satiety via capsaicin-sensitive neurons. The results suggest the possibility that more than one mechanism may mediate the effects of BBS: a neural mechanism involved in consummatory responses and a humoral mechanism involved in the operant response.     

Immune response to phosphorylcholine. VII. Functional evidence for three separate B cell subpopulations responding to TI and TD PC-antigens

A T-independent PC antigen, the C-polysaccharide, was used to induce a primary response to PC in splenic foci containing precursors taken from normal, unprimed BALB/c mice. The precursor frequency for the TI response was compared with the frequency for a T-dependent PC antigen, PPC-TGG-Hy. Idiotype analysis of the precursors for TD and TI responses indicate that the TD responses are more diverse with respect to clonal dominance of idiotype, TEPC-15, than that of TI responses. If splenic fragments were doubly immunized with both TI and TD antigens, the combined PC-specific precursor frequency was superadditive. TD-antigen-induced responses were found resistant to tolerogen and anti-idiotype suppression, whereas TI responses were extremely sensitive to both manipulations. Since under limiting dilution conditions precursors sort out independently, the superadditive response seen in dual immunized fragments can only be interpreted as evidence for an additional subpopulation of B cells that responds to the combined signals of TD and TI antigens. This postulated third B cell population is also extremely sensitive to tolerogen and anti-idiotype suppression.     

The allogeneic effect: the mechanism of allosuppression by Lyt-1, Ia- T cells

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.     

Mechanisms of tumor-induced immunosuppression: evidence for contact-dependent T cell suppression by monocytes.

BACKGROUND: The progressive growth of tumors in mice is accompanied by down-regulation of specific T cell responses. The factors involved in this suppression are not completely understood. Here, we have developed a model to examine the role of host immune effector cells in the inhibition of T cell function. In this model, progressive growth of a colon carcinoma line, CT26, is accompanied by loss of T cell response to alloantigens in both cytolytic and proliferation assays. MATERIALS AND METHODS: The CT26 tumor was inoculated into BALB/c syngeneic mice. Tumor growth, cytolytic T cell responses, lymphocyte proliferation, and flow cytometric analysis was performed in tumor-bearing animals 7 or 28 days after tumor inoculation. RESULTS: Spleen cells from tumor-bearing mice were found to suppress the proliferative response of spleen cells from normal mice to alloantigens. Examination of the spleen cell population by FACS analysis revealed an increase in the percentage of monocytes as defined by expression of CD11b, the Mac-1 antigen. Removal of the Mac-1-positive cells from the tumor-bearing hosts spleen relieved suppression of the tumor-bearing mouse spleen cell proliferative response to alloantigens, and addition of the Mac-1-positive enriched cells suppressed proliferation of normal T cells in response to alloantigens. Cell contact was required for this inhibition. CONCLUSIONS: Tumor induction of suppressive monocytes plays an important role in the general immunosuppression noted in animals bearing CT26 tumors. Identification of the mechanisms responsible for this effect and reversal of tumor-induced macrophage suppression may facilitate efforts to develop effective immunotherapy for malignancy.     

Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture

Addition of thyrotropin releasing hormone (TRH) to the medium of 2 clonal strains of functional rat pituitary cells stimulated the production of prolactin and inhibited growth hormone production. There was no effect on cell growth. Stimulation of prolactin production by TRH was detected within 4 hr, it reached a maximum level (2–5 times control) at 24–48 hr and persisted for at least 20 days in the continued presence of TRH. Stimulation was observed with a concentration of TRH as low as 0.10 ng/ml.     

T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.     

Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells

When Xenopus embryos are cultured in calcium- and magnesium-free medium (CMFM), the blastomeres lose adhesion but continue dividing to form a loose heap of cells. If divalent cations are restored at the early gastrula stage the cells re-adhere and eventually form muscle (a mesodermal cell type) as well as epidermis. If, however, the cells are dispersed during culture in CMFM, muscle does not form following reaggregation although epidermis does. This suggests that culturing blastomeres in a heap allows the transmission of mesoderm-induction signals from cell to cell while dispersion effectively dilutes the signal. In this paper, we have attempted to substitute for cell proximity by culturing dispersed blastomeres in XTC mesoderm-inducing factor (MIF). We find that dispersed cells do not respond to XTC-MIF by forming mesodermal cell types after reaggregation, but the factor does inhibit epidermal differentiation. One interpretation of this observation is that an early stage in mesoderm induction is the suppression of epidermal differentiation and that formation of mesoderm may require contact-mediated signals that are produced in response to XTC-MIF. We have gone on to study the suppression of epidermal differentiation in more detail. We find that this is a dose-dependent phenomenon that can occur in single cells in the absence of cell division. Animal pole blastomeres become more difficult to divert from epidermal differentiation at later stages of development and by stage 12 they are 'determined' to this fate. Fibroblast growth factor (FGF) also suppresses epidermal differentiation in isolated animal pole blastomeres and transforming growth factor-beta 1 acts synergistically with FGF in doing so.     

Enhancement and suppression of immunoglobulin G-producing B cells in the presence of immune T cells.

Mice were immunized one to three times with sheep red blood cells. Four to seven days after the last immunization, the spleens were removed and the cells were cultured in vitro in the absence of antigen. Removal of most T cells by anti-θ serum treatment prior to culture could increase the number of IgG-producing B cells without affecting the number of specific or nonspecific IgM-producing B cells detected after 2 days of culture. Addition of graded numbers of immune cells to pure immune B cells enhanced the number of IgG-producing B cells, whereas addition or higher number of immune cells caused suppression. Since removal of T cells could also enhance the proliferation of IgG-producing B cells induced by lipopolysaccharide (LPS), a polyclonal B-cell activator, it is suggested that the suppressive effects of high numbers of immune T cells are exerted directly on the B cells.     

Mechanism of electroporative dye uptake by mouse B cells.

The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.     

Mechanism of avian estrogen-induced hypertriglyceridemia: evidence for overproduction of triglyceride.

Relying on methods other than the determination of turnover rate of triglyceride from the curve of plasma triglyceride radioactivity after administration of labeled precursor, we have confirmed that the endogenous hypertriglyceridemia induced by estrogenization of the chick is accompanied by increased production of triglyceride. Chicks estrogenized with diethylstilbestrol became grossly hypertriglyceridemic and had elevated levels of plasma free fatty acid. Within 5 min of administration of labeled palmitate, estrogenized hypertriglyceridemic birds converted approximately 10 times more plasma free fatty acid to hepatic triglyceride than did controls. In addition, 2 hr after intraperitoneal injection of [14-C]acetate or [U-14-C]glucose, the specific activity of very low density lipoprotein triglyceride (VLDL-TG) of estrogenized birds reached or exceeded that of the untreated controls, and the rapid enrichment of the vastly expanded plasma VLDL-TG pool with labeled triglyceride further indicated that increased production of triglyceride occurs with estrogenization. Furthermore, [14-C]acetate incorporation into VLDL-TG was calculated to be 1.6 and 6.6% of the injected dose in estrogenized birds compared with 0.1 and 0.2% in untreated birds. Increased production of plasma VLDL-TG was confirmed by a kinetic study of VLDL-TG metabolism, employing reinjected, endogenously prepared [14-C]triglyceride-labeled VLDL. The fractional turnover rate of VLDL-TG in estrogenized hypertriglyceridemic birds was substantially less than that in untreated controls (0.32 plus or minus 0.03 vs 0.71 plus or minus 0.03/hr), but the total turnover rate was nearly 50 times greater (244 plus or minus 52 vs. 5 plus or minus 1 mg/hr).     

Characterization of a soluble suppressor of human B cell function produced by a continuous human suppressor T cell line. II. Evidence for suppression through a direct action of CTC-SISS-B on human B cells

CTC-SISS-B is an antigen-nonspecific suppressive lymphokine elaborated by an interleukin 2-dependent suppressor T cell line that produces noncytotoxic inhibition of human B cell but not T cell function. Like SISS-B, a soluble suppressive lymphokine present in the supernatants of Con A-activated peripheral blood T cell cultures, CTC-SISS-B is of 60,000 to 90,000 m.w., and its action is blocked by the simple sugar L-rhamnose. CTC-SISS-B inhibits human B cell Ig production and proliferation through a direct interaction with human B cells rather than through indirect effects on immunoregulatory T cells or monocytes. CTC-SISS-B suppression occurs through inhibition of an early event(s) in B cell activation since proliferation and Ig production by established human B cell lines are not inhibited by this lymphokine. Despite sharing many biochemical and biologic properties, CTC-SISS-B and gamma-interferon appear to be distinct mediators.     

Saturable attachment sites for polyhedron-derived baculovirus on insect cells and evidence for entry via direct membrane fusion.

This research provides the first evidence for specific receptor binding of polyhedron-derived baculovirus (PDV) to host cells and to lepidopteran brush border membrane vesicles (BBMV) and demonstration of entry via a nonendocytotic pathway involving direct membrane fusion. The technique of fluorescence-activated cell sorting analysis was used to investigate the specificity of binding between the PDV phenotype of Lymantria dispar nuclear polyhedrosis virus (LdNPV) and host membranes. Fluorescein isothiocyanate-labeled PDV was found to bind in a saturable manner to the gypsy moth cell line IPLB-LdEIta and to L. dispar BBMV. The IPLB-LdEIta cell line was found to possess approximately 10(6) PDV-specific receptor sites per cell. Excess levels of unlabeled PDV were highly efficient in competing with fluorescein isothiocyanate-labeled PDV for limited receptor sites, further supporting the specificity of the interaction. Major reductions in virus binding (as high as 70%) after protease treatment of cells indicated that a protein receptor is involved. A fluorescence dequenching assay of membrane fusion with octadecyl rhodamine B (R18)-labeled PDV was used to identify PDV fusion to host cells and BBMV. Direct membrane fusion of PDV occurred at 27 degrees C to both target membranes as well as at 4 degrees C at approximately 55% of the levels achieved at 27 degrees C. Viral fusion to BBMV occurred throughout the pH range of 4 to 11, with dramatically increased fusion levels (threefold) under the alkaline conditions normal for lepidopteran larval midguts. Treatment of cells with chloroquine, a lysosomotropic agent, did not significantly affect PDV fusion to cells or infectivity in tissue culture assays.     

Facilitated antigen presentation by B cells expressing IgD when responding T cells express IgD-receptors

In vitro studies have confirmed that cognate interactions between T and B cells are required to demonstrate enhanced helper activity using T cells with upregulated IgD-receptors (IgD-Rs). We studied the mechanism by which IgD-R+ T cells facilitate antibody responses by examining whether T cells also benefit from their expression of IgD-R. Experiments were designed to determine whether upregulation of IgD-R on T cells facilitates antigen presentation by IgD+ B cells. Goat Ig-primed splenic T cells from BALB/c mice were tested for their ability to respond to antigen-presenting B cells treated with goat anti-mouse (GAM) IgM or GAM IgD. T cell responses to GAM IgM and GAM IgD presented by B cells were significantly higher when goat Ig-primed cells were induced to express IgD-R by exposure to oligomeric IgD compared with goat Ig-primed control T cells. This effect was inhibited when monomeric IgD was added to the cultures. No differences in T and IgD-R+ T cell responses were seen using adherent cells as APCs. B cells from IgD-/- mice were also tested. Such B cells present antigen to IgD-R+ T cells without promoting enhanced responses compared with B cells from heterozygous IgD+/- mice. These studies suggest that IgD may play a costimulatory role during antigen presentation. We conclude that when T cells are induced to express IgD-R, these lectin-like receptors can ligate B cell membrane IgD during antigen presentation to facilitate responses of each of the cells engaged in cognate interaction, yielding enhanced antigen-specific T cell and B cell responses.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.     

Mechanism of mastocytoma-mediated suppression of lymphocyte reactivity.

Previously we have shown that mitomycin-treated P-815 (H-2d) cells (P-815m) inhibit the in vitro response of C57BL/6 spleen cells to mitogens and DBA/2 alloantigens. The present data indicate that P-815m cells also inhibit the response of C57BL/6 spleen cells to AKR (H-2k)-stimulating cells and that the inhibition does not appear to be the result of crowding. Subpopulations of spleen cells obtained by density gradient centrifucation or after renoval of glass-adherent cells are all sensitive to the inhibitory effects of P-815m. We also show that P-815m cells appears to operate both in vitro and in vivo. The experiments characterize the suppression of MLC reactivity by cell-free preparations of sonicated P-815 mastocytoma cells. The results suggest that the inhibition is not removed by ultracentrifugation, ultra-violet irradiation, or dialysis of the sonicate. P-815 sonicates subjected to chloroform extraction, heating at 56 degrees C, 0.1 mu filtration, or treatment with anit-minute virus of mice serum are still inhibitory. This inhibition is resistant to RNAase treatment but destroyed by pronase. P-815 sonicate suppresses the response of C57BL/6 spleen cells to both DBA/2 and AKR-stimulating spleen. Inhibition of MLC reactivity is only seen if P-815 sonicate is added within the first 48 hr after the initiation of culture. These results indicate that the inhibition of MLC by sonicates of P-815 cells is due to a nondialyzable protein whose effects are not H-2 restricted and are acting on the early phase of sensitization.