Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

Reaginic antibody formation in the mouse. VI. Suppression of IgE and IgG antibody responses to ovalbumin following the administration of high dose urea-denatured antigen.

The major antigenic determinants in ovalbumin molecules (OA) were lost following denaturation in 8 M urea. The urea-denatured antigen (UD-OA) failed to combine with anti-OA antibody, but was capable of priming mouse T cells specific for OA. BDF₁ mice primed with alum-precipitated OA were given three intravenous injections of 100 μg UD-OA at 3, 5, and 7 days after the primary immunization. The treatment with UD-OA suppressed both IgE and IgG antibody responses to OA. The same treatment of OA-primed animals with intravenous injections of OA resulted in suppression of IgE antibody response but enhanced IgG antibody response. Intravenous injections of either OA or UD-OA suppressed both IgE and IgG anti-DNP antibody responses of DNP-OA-primed animals but failed to suppress anti-hapten antibody responses to DNP-keyhole limpet hemocyanin. The effect of the treatment on helper T cells and B memory cells in OA-primed animals was studied by adoptive transfer experiments. The results showed that the OA-treatment as well as UD-OA-treatment suppressed the development of both helper function of T cells and B memory cells in the spleen, but the UD-OA treatment was more effective than OA-treatment for the suppression of B cell development. Possible mechanisms for the suppression of the development of immunocompetent cells were discussed.     

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.     

Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent

The experimental conditions for the preparation of conjugates of ovalbumin (OA) and monomethoxypolyethylene glycol (mPEG) of a preselected average degree of conjugation, n, using cyanuric chloride as the coupling agent, have been investigated with emphasis on purification and characterization of the products. These conjugates served as prototypes of tolerogenic mPEG derivatives of antigenic proteins which were capable of suppressing in mammals the immunological response to the corresponding unmodified antigens. In other studies in this laboratory, the tolerogenicity of OA(mPEG)n conjugates was found to be a function of n. The reproducibility of the reaction leading to the production of OA(mPEG)n conjugates was shown to depend primarily on the reactivity of the mPEG-cyanuric chloride intermediate, which--for best results--had to be synthesized under completely anhydrous conditions. Isolation of the OA(mPEG)n conjugates was optimized by the use of ion-exchange chromatography whereby rapid removal of large amounts of uncoupled intermediate from the conjugate was achieved; the conditions of fractionation were affected by the degree of conjugation. This method of purification was superior to dialysis, ultrafiltration, and gel filtration. Furthermore, by the application of analytical hydrophobic interaction HPLC it was possible to differentiate among conjugates of different degrees of conjugation and to establish the absence of any detectable free OA in any of the preparations. The quantity of mPEG in the conjugates was determined directly by NMR.     

Differential abilities of human peripheral blood monocytes quantitatively or qualitatively differing in HLA-DR and HLA-DS expression to support B cell activation in liquid and semisolid cultures

The present investigation was performed to determine whether the activation of human B cells by Staphylococcal protein A (SpA) in liquid and semi-solid cultures might be dependent on distinct subsets of peripheral blood mononuclear-phagocytes (M phi) defined by the expression of HLA-DR and HLA-DS determinants. Highly pure HLA-DR- M phi functioned as effectively as HLA-DR+ MO in supporting B cell liquid proliferative responses when SpA was continuously present in cultures. However, HLA-DR+ M phi were two to three times more effective than HLA-DR- M phi in promoting B cell proliferative responses when either M phi or B cells were pulsed with SpA and were then cultured without supplemental SpA. Similarly, B cell activation in semisolid cultures was crucially dependent on HLA-DR+ M phi because colony responses were reduced fivefold in the presence of M phi expressing low/intermediate HLA-DR levels compared to M phi-containing cells with high HLA-DR levels. HLA-DS- M phi isolated by two different techniques were more effective than HLA-DS+ M phi in supporting both liquid proliferative and colony responses of B cells. Flow microcytofluorometry analysis of the dual expression of HLA-DR and HLA-DS on highly pure HLA-DR- M phi and HLA-DR+ M phi revealed that both HLA-DR- and HLA-DR+ M phi expressed low levels of HLA-DS. Importantly, the expression of HLA-DS on HLA-DR- M phi was bimodal, with an HLA-DR-, DS+ subset and an HLA-DR-, DS-subset being present. Other experiments supported the conclusions that the differential abilities of the HLA-DR-, -DS-defined subsets of M phi to support B cell activation did not represent M phi suppressive effects or differences in IL 1 production. Collectively, these results indicate that B cell activation can be directly supported by M phi whose predominant phenotype is HLA-DR+, -DS-. Thus, the accessory cell pathway of B cell activation described here is distinct from the pathway known to be required for T cell responsiveness, and could serve to provide early alternative or ancillary signals for triggering B cells.     

DNP-phycobiliproteins, fluorescent antigens to study dynamic properties of antigen-IgE-receptor complexes on RBL-2H3 rat mast cells

In RBL-2H3 rat mucosal mast cells, the crosslinking of cell-surface IgE-receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNP)-conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B-phycoerythrin and C-phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. The binding of these antigens to IgE-receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 microgram/ml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately 26 X 10(3) molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigen-stimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP-lysine, is added to degranulating cell suspensions. DNP-lysine also displaces surface-bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP-lysine to displace surface-bound DNP42-B-phycoerythrin from IgE-receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen-stimulated F-actin assembly enhances secretion and delays the transition of antigen to its DNP-lysine-resistant form. Cytochalasin treatment also permits the long-range movement of antigen into surface caps.(ABSTRACT TRUNCATED AT 250 WORDS)     

T cell specificity in twice-irradiated F1----parent bone marrow chimeras: failure to detect a role for immigrant marrow-derived cells in imprinting intrathymic H-2 restriction

In an attempt to resolve the issue of whether H-2-restricted T cell specificity is controlled by thymic epithelial cells or by cells of the macrophage/dendritic cell (M phi/DC) lineages, long-term F1----parent chimeras were subjected to secondary irradiation and reconstitution with F1 marrow cells. The rationale was that if F1 M phi/DC enter the thymus only quite slowly after irradiation, as claimed by other investigators, leaving F1----parent chimeras for a period of several months before re-irradiation would ensure that the new wave of T cells generated in the thymus of the chimeras would have no difficulty in making contact with donor-derived F1 M phi/DC. According to the view that M phi/DC rather than epithelial cells control H-2 restriction, the T cells differentiating in these chimeras would be expected to show H-2 restriction to both parental strains. In practice, T cells from twice-irradiated (1000 + 800 rad) chimeras showed strong restriction to host (thymic) H-2 determinants, the degree of restriction to host determinants being as marked as with T cells from once-irradiated chimeras. This finding applied both to T proliferative responses to KLH assayed in vitro and to T helper function for sheep erythrocytes measured in vivo. Preliminary experiments established that the initial dose of irradiation used for preparing the chimeras (1000 rad) resulted in almost total replacement of intrathymic M phi/DC by donor-derived cells within 4 wk of irradiation; M phi/DC were typed by determining their capacity to stimulate mixed-lymphocyte reactions. Collectively, the data imply that, at least under the conditions used, H-2-restricted T cell specificity is controlled by epithelial cells rather than by M phi/DC.     

Reaginic antibody formation in the mouse. VII. Induction of suppressor T cells for IgE and IgG antibody responses.

Intravenous injections of urea-denatured ovalbumin (UD-OA) into OA-primed high responder mice suppressed the antibody response not only to the priming antigen but also to subsequent immunization with dinitrophenyl derivatives of OA (DNP-OA). The transfer of normal spleen cells or OA-primed spleen cells into UD-OA-treated animals did not restore the capacity of responding to DNP-OA to form anti-DNP IgE and IgG antibodies. The transfer of splenic T cell fraction from the UD-OA-treated animals into normal syngeneic mice diminished both IgE and IgG antibody responses of the recipients to DNP-OA. The B cell-rich fraction from the same donors failed to affect the anti-hapten antibody response and enhanced anti-cancer (OA) IgG antibody response of the recipients. It was also found that the transfer of T cell-rich fraction of OA-primed spleen cells failed to suppress antibody response of the recipients to DNP-OA. The results indicated that spleen cells of UD-OA-treated mice contained suppressor T cells which are distinct from helper cells. Suppressive activity of T cells in the UD-OA treated animals was specific for OA. The transfer of the T cell-rich fraction failed to suppress anti-DNP antibody response of the recipients to DNP-KLH.     

Hapten-specific IgE antibody responses in mice. VII. Conversion of IgE "non-responder" strains to IgE "responders" by elimination of suppressor T cell activity.

Mice of the inbred strains SJL (H-2s) and AKR (H-2k) are "non-responders" and "low-responders," respectively, in terms of their capacity to develop antibody responses of the IgE class when immunized with conventional proteins and hapten-protein conjugates under conditions optimal for eliciting IgE responses in "high-responder" mice, such as BALB/c (H-2d), to these same antigens. For example, BALB/c mice preimmunized with ASC and then challenged 7 days later with DNP-ASC develop peak augmented primary IgE anti-DNP antibody responses of 320 PCA units, whereas SJL and AKR mice develop responses which are 16-fold and 4-fold lower, respectively. However, pretreatment of the latter two strains with appropriate doses of either x-irradiation (150 R), cyclophosphamide (100 mg/kg) or ALS (150 mul) before carrier-preimmunization strikingly enhances the magnitude of IgE antibody responses in such mice to levels as high as 64-fold above those of untreated control mice of the same strains. Evidence obtained in these experiments indicates that the capacity of such maneuvers to to convert poor IgE responders to high responder status reflects elimination of nonantigen-specific suppressor T lymphocytes which are naturally present and normally function to suppress or "dampen" the IgE antibody response in a relatively selective manner. It appears that these cells modulate IgE responses by acting at least at two distinct points: 1) The most effective activity seems to be at the level of induction of carrier-specific helper T cells; 2) A second locus of inhibitory activity is more distal in the response, either impeding helper T cell-B cell cooperative interactions or suppressing B cell differentiation and/or function directly. Taken collectively, these observations demonstrate that the state of poor responsiveness of the SJL and AKR strains for the IgE antibody class is not a reflection of a genetic inability to develop IgE responses but rather a manifestation of a genetic capability to actively inhibit IgE antibody synthesis.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Regulation of the murine IgE antibody response. I. Characterization of suppressor cells regulating both persistent and transient responses and their sensitivity to low doses of X-irradiation.

Anti-ovalbumin (OA) IgE antibody responses were measured in B6D2F1 mice as a function of time and antigen dose. One hundred to 200 microgram of OA in Al(OH)3 elicited transient responses, whereas 1 to 10 microgram of OA in Al(OH)3 elicited persistent anti-OA IgE responses of high titer. T cells isolated from the spleens of mice mounting either a persistent or a transient response strongly suppressed primary anti-DNP IgE responses in unirradiated recipient mice that were immunized with DNP-OA in Al(OH)3; it was, therefore, concluded that suppressor T cells (Ts cells) were activated during both the persistent and transient IgE responses. Nevertheless, in the present study it was not possible to completely rule out the contention that IgG antibodies may also have been suppressing the IgE response. With a modified adoptive transfer system, it was shown that these Ts cells were sensitive to low doses (250 R) of x-irradiation. The suppressive activity of long-term OA primed cells was also shown to be markedly enhanced when cultured for 24 hr with soluble OA; this finding was interpreted to indicate the presence of memory suppressor cells.     

Antigen concentration determines helper T cell subset participation in IgE antibody responses.

A recently developed in vitro system for antigen-stimulated primary and secondary murine IgE antibody responses has been used to define (a) the relative participation of the Th1 and Th2 cell-derived lymphokines IFN-gamma and IL-4, respectively, in such responses, and (b) the role of antigen concentration in determining functional helper T cell activity. These studies confirm that IL-4 and IFN-gamma exert regulatory effects on IgE synthesis, but the nature and extent of their respective effects on primary and secondary IgE responses differ. Thus, primary IgE responses are considerably more sensitive to and dependent on IL-4 than are secondary IgE responses since (1) anti-IL-4 monoclonal antibody totally inhibited primary IgE responses, but only partially affected secondary responses; and (2) exogenously added IL-4 could stimulate primary IgE responses to optimal antigen concentrations, but had no effect on secondary IgE production. Likewise, antigen-stimulated primary IgE responses are about eightfold more sensitive than are secondary responses to the inhibitory effects of IFN-gamma. Studying the effect of antigen dose on the quantity of IgE antibody produced revealed that although IFN-gamma could be detected by ELISA in cultures exhibiting high-dose antigen-dependent diminution of IgE production, anti-IFN-gamma monoclonal antibody could not reverse this phenomenon. Thus, IFN-gamma is not solely responsible for decreased IgE synthesis associated with high-dose antigen exposure. IL-4 activity was detected in the fluid from cultures stimulated with low, but not high, levels of antigen. Moreover, addition of exogenous IL-4 restored IgE production to normal levels in cultures exposed to high antigen concentrations. Therefore, it appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells. These results help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.     

Immunologic tolerance to allergenic protein determinants: properties of tolerance induced in mice treated with conjugates of protein and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL).

Conjugates of proteins and the synthetic copolymer of D-glutamic acid and D-lysine (protein-D-GL) reproducibly induce significant unresponsiveness to the protein antigens in experimental mice. Proteins studied include ovalbumin and antigen E of ragweed extract, the major allergen of ragweed pollen. The unresponsive state 1) can be induced in both unsensitized and previously sensitized experimental animals, 2) is selectively confined to responses of the IgE antibody class, 3) is long lasting, and 4) is highly antigen specific. IgE antibody responses can also be suppressed by administering comparable doses of unconjugated protein alone; however, the unresponsive state induced in this manner is only transient and rebound production of IgE antibody is often observed. Results from the studies of the cellular basis of the protein-D-GL induced unresponsiveness demonstrate that 1) protein-D-GL conjugates do not induce unresponsiveness at the level of protein-specific B cells, 2) tolerance is not induced by virtue of a detectable antigen-specific suppressor T cell mechanism, 3) tolerance is most probably induced in the antigen-specific helper T cell populations. The significant IgE-selective and antigen-specific tolerogenic activity of protein-D-GL conjugates make these compounds potential candidates for use as therapeutic agents in the treatment of IgE-mediated human allergic disorders induced by protein allergens.     

A requirement for physical linkage between determinants recognized by helper molecules and cytotoxic T cell precursors in the induction of cytotoxic T cell responses

It has long been understood that both antibody and delayed-type hypersensitivity responses are induced through collaborative events in which the determinants recognized by the precursor cells must be physically linked to the determinants recognized by the helper. Although it is clear that the generation of memory cytotoxic T lymphocyte precursors (CTLp) involves linked recognition of determinants, the induction of CTL responses has been viewed as being dependent upon interleukin 2 (IL 2), which could be provided by a helper cell, but independent of requirements for antigen bridging. In this work, we have designed a system that lacks exogenous IL 2 by using as our source of help, antigen-specific helper molecules derived from helper T cells. These soluble helper molecules are uncontaminated by IL 2 and unlike a helper cell, are unable to produce IL 2. Helper molecules specific for chicken red blood cells (Crbc) and for a synthetic polypeptide, poly 18, were tested. Thymocyte responders require a source of help to respond to alloantigens intrinsically expressed on the surface of adherent stimulator cells. To analyze the mechanism whereby the helper molecules acted, we used a system involving recognition of haptenic and carrier determinants that were physically linked by virtue of being located on the same cell surface (intra-structural linkage). Adherent stimulator cells were pulsed with Crbc or poly 18 so that the alloantigens recognized by the thymocyte CTLp (intrinsically expressed class I) were either linked or unlinked to the carrier determinants (Crbc or poly 18) presented by the adherent cells and recognized by the helper molecules. Both types of helper molecule were shown to be antigen-specific in crisscross experiments. The helper molecules specific for Crbc were able to induce the thymocyte CTLp only when both hapten and carrier were present on the same stimulator cell surface. Because we were not able to detect a requirement for H-2-restricted recognition of carrier antigen, this inductive event must be viewed as requiring linked associative recognition of determinants, but being noncognate. In contrast, the helper molecules recognizing poly 18 showed a requirement for both physical linkage of determinants and for H-2 restricted recognition, indicating that the mechanism of induction was cognate in nature. Therefore, we have shown that interactions between CTLp and soluble, antigen-specific, helper cell-derived inductive molecules are similar in nature to those of other T cell precursors and of B cells in the stringent requirement for close physical proximity achieved by linked or cognate recognition of determinants across an antigen bridge.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of nominal antigen and Ia antigen in the binding of antigen-specific T lymphocytes to macrophages.

We have previously demonstrated that when primed T lymphocytes were repeatedly incubated on monolayers of antigen-pulsed macrophages (M phi), the cells that failed to adhere to the monolayer demonstrated a marked depletion of their proliferative response that was specific both for the antigen used for pulsing the M phi and for Ia determinants on the M phi. In order to further analyze the contribution of the nominal antigen and Ia antigens to the physical binding of T lymphocytes to M phi, we have attempted to block the absorption of T lymphocytes to M phi with a large excess of soluble antigen and with anti-Ia sera. Our results demonstrate that anti-Ia sera inhibit but that soluble antigen augments the binding of specific T lymphocytes to M phi. The implications of these findings for "dual recognition" and "linked recognition" models of T lymphocyte receptors are discussed.     

A novel role for macrophages: the ability of macrophages to tolerize B cells

We investigated the ability of macrophages (M phi) to present the tolerogen fluoresceinated sheep gamma-globulin (FL-SGG) to B cells. M phi pulsed with FL-SGG or murine FL-IgG2 were able to tolerize normal spleen B cells specifically as assessed by the plaque-forming cell (PFC) response to the antigens FL-Brucella abortus (FL-BrA) and FL-polymerized flagellin (FL-POL). Tolerance was not induced when M phi were pulsed with a variety of other FL antigens or the synthetic tolerogen FL-D-glutamic acid-D-lysine (FL-D[G,L]). The ability of M phi to tolerize B cells was not T cell-dependent, because populations of both T-depleted B cells and hapten-specific B cells were tolerizable. M phi-induced B cell tolerance did not exhibit genetic restriction with regard to the H-2 haplotype of the presenting M phi or the responding B cells. A variety of different types of peritoneal M phi, including normal resident M phi and those elicited by thioglycollate or concanavalin A (the latter are predominantly la+), could tolerize B cells after being pulsed with FL-SGG. We compared tolerogen-pulsed M phi to soluble tolerogen for the ability to tolerize B cells and found that tolerogen bound to M phi was more than 10 times as potent as an equivalent amount of soluble tolerogen. In contrast to the ability of M phi to present FL-SGG in a tolerogenic fashion to B cells, the P388AD lymphoid dendritic cell-like tumor line presented FL-SGG in an immunogenic mode to B cells. Tolerogen bound to P388AD cells could specifically augment a PFC response to both FL-BrA and FL-POL. We suggest that certain types of M phi may play a role in unresponsiveness by enhancing the tolerogenicity of soluble antigen, whereas other accessory cells may present the same moieties in an immunogenic fashion.     

Regulation of antibody response in different immunoglobulin classes. II. Induction of in vitro IgE antibody response in murine spleen cells and demonstration of a possible involvement of distinct T-helper cells in IgE and IgG antibody responses.

In vitro induction of anti-DNP IgE as well as IgG1, IgG2a antibody responses was shown in murine spleen cell culture. Spleen cells primed three times with 1 mug of DNP-OA or DNP-Asc produced significant amounts of anti-DNP IgE as well as IgG antibodies by the in vitro stimulation with DNP-OA or DNP-Asc, respectively. Collaboration between DNP-primed B cells and carrier-primed T cells was required for the induction of both IgE and IgG antibodies with DNP-coupled T-dependent antigen. Carrier-specific T cells induced with a low dose of Asc (0.01 mug) showed helper function only on IgE antibody response, whereas T cells primed with a higher dose of Asc (10 mug) cooperated only with IgG-B cells. T cells primed with Asc in CFA showed helper function mainly on IgG antibody response but not on IgE antibody response. The result indicated the presence of a distinct population of T helper cells for IgE and IgG antibody responses. T-independent antigen (DNP-Ficoll) induced both anti-DNP IgE and IgG antibody responses in DNP-primed spleen cell population without the requirement of the collaboration of helper T cells.     

Enhancement of antigen-presenting function of dendritic cells with culture supernatants of mouse peritoneal macrophages stimulated with certain particulate substances

The production from murine resident peritoneal macrophages (M phi) of a soluble factor, which was capable of enhancing the antigen-presenting (AP) function of dendritic cells (DC), was examined. The supernatants of peritoneal M phi (M phi sup) were prepared by culturing peritoneal M phi with particles, i.e., zymosan A, latex, and sheep red blood cells (SRBC), or antigen-antibody (Ag-Ab) complexes such as keyhole limpet hemocyanin (KLH)-anti-KLH, ovalbumin (OVA)-anti-OVA, and SRBC-anti-SRBC complexes. When exposed to M phi sup during antigen pulsing DC induced a marked antigen-specific T cell proliferation, relative to DC treated with the supernatants from M phi cultured without stimuli (control sup). On the other hand, M phi sup-treated splenic M phi stimulated antigen-specific T cell activation to almost the same extent as did splenic M phi treated with control sup. These results indicated that peritoneal M phi elaborated a soluble factor which preferentially enhanced the AP capacity of DC when stimulated with particles or Ag-Ab complexes. Analytical gel filtration of M phi sup revealed that the factor had an apparent molecular weight of 27,000 daltons which was distinct from interleukin 1.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.