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Clinical Research

Proc Natl Acad Sci U S A

T cell tolerance can be induced by B cell presentation of Ags to naive T cells. To further characterize this mechanism of T cell tolerance induction, we have investigated the effects of injecting mice with an intact rat IgG2a Ab, which binds to the B cell low-affinity Fc epsilon receptor (CD23), on the responsiveness of B cells and T cells to rat IgG2a. Our observations indicate that 1) intravenous, subcutaneous, or intraperitoneal injection of this Ab induces antigen-specific B cell and T cell tolerance; 2) both forms of tolerance are induced more completely by injection of rat IgG2a anti-CD23 mAb than by injection of an equal dose of a control rat IgG2a Ig; and 3) reduced responsiveness to Ag is seen as early as 1 to 3 days after anti-CD23 mAb injection and reaches maximum levels by 7 days after injection. Although tolerance induced by the injection of soluble proteins has been reported to be characterized by reduced production of IL-2 and IFN-gamma, but normal production of IL-4, injection of mice with rat IgG2a anti-mouse CD23 mAb greatly decreases the IL-4 response to a rat IgG2a immunogen that normally induces a large IL-4 response.

IL-4 has been shown to induce B lymphocytes to switch from the expression of membrane IgM to the expression of membrane IgE and to be required for the generation of primary polyclonal and secondary Ag-specific IgE responses in mice. To further define the role of IL-4 in the generation of memory IgE responses, we investigated the ability of a combination of anti-IL-4 and anti-IL-4R mAb to block the generation of secondary IgE responses induced by: 1) a second infection with the nematode parasites Nippostrongylus brasiliensis or Heligmosomoides polygyrus; or 2) injection of anti-IgD antibody-primed mice with anti-IgE antibody. The latter stimulus was designed to induce intrinsic membrane IgE-expressing B cells to differentiate into IgE-secreting cells. Although the IgE responses induced by a second nematode infection were completely inhibited by the combination of anti-IL-4 and anti-IL-4R mAb, anti-IgE antibody-induced IgE responses in anti-IgD primed mice were not inhibited by these antibodies to a large degree. Additional experiments demonstrated that the anti-IgE antibody-induced memory IgE response was dependent on CD4+ T cells but did not involve the low affinity B cell Fc epsilon RII. Taken together, these observations provide evidence that IL-4 is required for virgin B lymphocytes to develop into IgE-expressing cells, but is not required for B cells that express intrinsic membrane IgE to differentiate into IgE-secreting cells in a T-dependent response. Furthermore, these data suggest that secondary IgE responses in the parasite models that we have studied develop from B cells that had not previously switched to the expression of IgE.

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype. Injection of H delta a/1-BSA into BALB/c mice, which express Ig of the a allotype, or into (BALB/c x CB20)F1 mice (a x b allotype heterozygotes) induced IgG1 anti-BSA antibody responses that peaked 8 to 9 days after injection, and were more than 1000 times larger than those induced by injection of BSA alone, and 100 times larger than those induced by injecting unconjugated BSA plus H delta a/1. H delta a/1-BSA was no more immunogenic than unconjugated BSA when injected into CB20 mice, which express Ig of the b allotype, while AF3.33-BSA greatly enhanced anti-BSA antibody production in CB20, but not in BALB/c mice. Mice serially immunized with three different Ag conjugated to H delta a/1 made large antibody responses to all three Ag, provided that the mouse strain used did not recognize allotypic determinants on H delta a/1 as foreign and produce a neutralizing antibody response. Intravenous and s.c. routes of inoculation produced responses of similar magnitude and relatively low variability; responses to footpad or intramuscular inoculation were more variable, and i.p. inoculation induced smaller responses. Injection of BALB/c mice i.v. with 100 micrograms of H delta a/1-BSA induced an IgG1 anti-BSA response of 5.6 mg/ml, which was approximately 70% of the total IgG1 response. Anti-BSA responses to 30 micrograms of conjugate or less were much smaller, but could be considerably enhanced by adding unconjugated H delta a/1 to the inoculum. This system will be useful for the rapid stimulation of large antibody responses to biologically important Ag, and for investigating mechanisms of Ag processing and B and T cell activation.

A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype. These cells process the conjugate and present the foreign Ag to Ag-specific T lymphocytes, which become activated. Thus, B cells of the recognized allotype can be stimulated by cross-linking of their mIgD, Ag-specific T cell help, non-Ag-specific cytokines, and non-Ag-specific contact with activated T cells. In contrast, B cells that express the Igh allotype not recognized by the Ag-anti-IgD antibody conjugate (bystander B cells) can be stimulated in this system only by non-Ag-specific cytokines and non-Ag-specific contact with activated T cells. Although both recognized and bystander B cells in conjugate-injected mice demonstrated substantial increases in size and Ia expression, only the recognized B cells were induced to synthesize DNA and to make a substantial polyclonal Ig response. Bystander B cells still failed to secrete IgG when mice were injected with an anti-IgD-Ag conjugate specific for the other Igh allotype as well as a mAb that cross-linked IgD of the bystander B cell allotype. These observations demonstrate that although non-Ag-specific cytokine and contact-mediated T cell help are sufficient to induce B cells to increase in size and Ia expression in anti-IgD antibody-injected mice, Ag-specific T cell help is required to stimulate the generation of an IgG response in these mice.

Dendritic cells (DC) are unmatched among APCs in their ability to bind, process, and present Ag. Presentation by such potent APCs, if always immunogenic and never tolerogenic, might stimulate pathogenic autoimmune responses. To determine whether Ag presentation by DC can induce tolerance, mice were injected with a rat IgG2b anti-splenic DC mAb, 33D1, and challenged 13 to 28 days later with a stimulatory rat IgG2b mAb. Injection of mice with 1 ng/100 micrograms of 33D1 rarely induced an anti-rat IgG2b Ab response and, in most mice, induced rat IgG2b-specific T cell and B cell tolerance. Tolerant mice had decreased ability to secrete Ab and make both type 1 and type 2 cytokine mRNA and protein in response to immunization with rat IgG2b. 33D1 was 100- to 1000-fold more potent as a tolerogen than an isotype-matched control rat IgG2b mAb. Injecting mice with aggregated 33D1, 33D1 plus anti-IgD mAb, or 33D1 plus IL-1 induced an IgG1 anti-rat IgG2b Ab response rather than tolerance. IL-1 injected 3 days after 33D1 still induced an Ab response rather than tolerance. Not all anti-DC mAbs are tolerogenic. Injection of a DC-specific hamster anti-CD11c mAb (N418) stimulates an IgG anti-hamster response, and injection of 33D1 plus N418 stimulates both anti-hamster and anti-rat IgG2b responses. These observations indicate that DCs can present Ag in either a tolerogenic or stimulatory manner and suggest that inflammatory stimuli can convert an otherwise tolerogenic signal to a stimulatory signal.

A spleen cell population that lacks CD3, CD4, CD8, Thy-1, B220, and class II major histocompatibility complex cell-surface markers (non-B, non-T cells) produces IL-4 when cultured in wells coated with IgE. Their production of IL-4 in response to plate-bound (PB)-IgE is strikingly enhanced by IL-3, and in the presence of IL-3, these cells also produce IL-4 in response to PB-IgG2a. The effect of IL-3 is not mimicked by IL-1, IL-2, IL-5, IL-6, IL-7, granulocyte-macrophage CSF (GM-CSF) or IFN-gamma. Non-B, non-T cells cultured with IL-3 for 12 h acquire the capacity to produce enhanced amounts of IL-4 in response to subsequent culture with PB-Ig even if IL-3 is omitted from the second culture. Irradiated cells also respond to IL-3 with enhanced capacity to produce IL-4 to PB-Ig, indicating that cell proliferation is not required for the effect of IL-3. The IL-3 effect can be obtained in vivo; treatment of mice with a total dose 90,000 U of synthetic IL-3 over a 3-day period results in the presence of splenic and peritoneal cavity non-B, non-T cells that produce enhanced amounts of IL-4 in response to PB-Ig. The FcR that mediates the response to PB-IgE appears to be Fc epsilon RI because cells can be sensitized with IgE anti-DNP mAb, washed, cultured for 15 h at 37 degrees C, washed again, and stimulated to produce IL-4 with 0.1 to 1 ng/ml of TNP10-OVA. IL-3 does not appear to mediate its function by increasing the number of Fc epsilon RI because it can exert its effect when cultured with non-B, non-T cells after they have been sensitized with IgE anti-DNP. However, IL-3 pretreatment does affect the signaling process in that non-B, non-T cells sensitized with IgE anti-DNP show strikingly reduced production of IL-4 to concentrations of TNP10-OVA of 100 ng/ml or more whereas cells pretreated with IL-3 show little or no diminution in IL-4 production at concentrations of TNP10-OVA up to 1 microgram/ml.

To further define requirements for T cell help in the stimulation of an in vivo IgE response, we studied a system in which the injection of mice with a goat antibody to mouse IgD (GaMD) stimulates large polyclonal IgG1 and IgE responses. In this system, both responses are blocked by anti-CD4 antibody, but only the IgE response is blocked by (anti-IL-4) antibody. Anti-CD4 antibody, if injected 5 days after GaMD, was found to inhibit the GaMD-induced IgE response to a much greater extent than the IgG1 response, even though both responses occur simultaneously and are inhibited to an equal extent by optimal or suboptimal doses of anti-CD4 antibody administered 2 days after GaMD. Even a suboptimal, 50-micrograms dose of anti-CD4 antibody, when injected 5 days after GaMD, inhibited the IgE response to a much greater extent than did an optimal 10-mg dose of anti-IL-4 antibody injected at the same time, even though 10 mg of anti-IL-4 antibody more completely inhibited GaMD-induced IgE production than did 50 micrograms of anti-CD4 antibody when injected 2 days after GaMD. These observations provide evidence that a late acting form of T cell help other than IL-4 is important for the generation of an IgE response but not an IgG1 response in GaMD-immunized mice.

CD40/CD40 ligand interactions are required for the development of T cell-dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response.

In vivo experiments were performed to determine if the cross-linking of mlg on antigen-presenting B cells could induce them to present Ag to naive T cells in a stimulatory rather than a tolerogenic fashion. Mice were injected with a foreign mAb to a B cell Ag (Fc epsilon RII or CR1), and/or a self-anti-IgD mAb. Injection of either mAb alone failed to induce an Ab response; however, simultaneous injection of the foreign anti-B cell mAb plus the self-anti-IgD mAb stimulated a large response. Inasmuch as injection of the anti-IgD mAb should not have facilitated transfer of the foreign mAb to dendritic cells, this observation suggests that cross-linking of B cell mlg can induce B cells to acquire the ability to present Ag to naive T cells in an activating manner. Furthermore, when injected with anti-IgD mAb, both foreign anti-B cell mAbs were more potent in inducing Ab responses in this system than were isotype-matched control mAbs, consistent with the hypothesis that T cells were activated by Ag-presenting B cells. Results of dose-response and cell transfer studies, however, suggested that stringent cross-linking of B cell mlg on large numbers of B cells is required for B cell Ag presentation to induce T cell activation rather than tolerance. Therefore, these observations suggest that professional APCs usually are required to activate naive T cells, and that B cell Ag presentation can only activate naive T cells under unusual circumstances.

The costimulatory signal provided to T cells through CD28/CTLA-4 interactions is required for in vivo Th cell effector function associated with cytokine production. However, it is uncertain whether the two well-characterized ligands for these molecules, B7-1 and B7-2, differentially influence the consequent development of a type 1 or a type 2 primary response. We have examined the in vivo effects of blocking B7-1 and/or B7-2 ligand interactions on the type 2 mucosal immune response that follows oral infection of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of the combination of anti-B7-1 and anti-B7-2 Abs inhibited H. polygyrus-induced increases in serum IgG1 and IgE levels, the expansion of mesenteric lymph node (MLN) germinal centers, in situ CD4+ T cell expansion, elevated blood eosinophils, and increased intestinal mucosal mast cells. Similarly, both Abs blocked MLN and Peyer's patch cytokine gene expression and elevations in MLN T cell-derived IL-4 protein secretion. However, in the same experiments, administration of either anti-B7-1 or anti-B7-2 Abs alone had little effect on any of these parameters. T cell and B cell activation was also blocked by the combination of anti-B7-2 and a B7-1-specific mutant Y100F CTLA-4Ig construct. These results suggest that to the extent that anti-B7-1 and anti-B7-2 mAbs block B7 interactions, either B7-1 or B7-2 ligand interactions can provide the required costimulatory signals that lead to T cell effector function during a type 2 in vivo immune response.