IL-3 induces B7.2 (CD86) expression and costimulatory activity in human eosinophils.

Eosinophils in tissues are often present in intimate contact with T cells in allergic and parasitic diseases. Resting eosinophils do not express MHC class II proteins or costimulatory B7 molecules and fail to induce proliferation of T cells to Ags. IL-5 and GM-CSF induce MHC class II and B7 expression on eosinophils and have been reported in some studies to induce eosinophils to present Ag to T cells. The cytokine IL-3, like IL-5 and GM-CSF, is a survival and activating factor for eosinophils and the IL-3 receptor shares with the IL-5 and GM-CSF receptors a common signal transducing beta-chain. IL-3-treated eosinophils expressed HLA-DR and B7.2, but not B7.1 on their surface and supported T cell proliferation in response to the superantigen toxic shock syndrome toxin 1, as well as the proliferation of HLA-DR-restricted tetanus toxoid (TT) and influenza hemagglutinin-specific T cell clones to antigenic peptides. This was inhibited by anti-B7.2 mAb. In contrast, IL-3-treated eosinophils were unable to present native TT Ag to either resting or TT-specific cloned T cells. In parallel experiments, eosinophils treated with IL-5 or GM-CSF were also found to present superantigen and antigenic peptides, but not native Ag, to T cells. These results suggest that eosinophils are deficient in Ag processing and that this deficiency is not overcome by cytokines that signal via the beta-chain. Nevertheless, our findings suggest that eosinophils activated by IL-3 may contribute to T cell activation in allergic and parasitic diseases by presenting superantigens and peptides to T cells.

Glucocorticoids upregulate CD40 ligand expression and induce CD40L-dependent immunoglobulin isotype switching.

IL-4 and CD40 ligation are essential for IgE synthesis by B cells. We have shown previously that hydrocortisone (HC) induces IgE synthesis in IL-4-stimulated human B cells. In this study we demonstrate that HC fails to induce IgE synthesis in B cells from CD40 ligand-deficient (CD40L-deficient) patients. Disruption of CD40L-CD40 interactions by soluble CD40-Ig fusion protein or anti-CD40L mAb blocked the capacity of HC to induce IgE synthesis in normal B cells. HC upregulated CD40L mRNA expression in PBMCs and surface expression of CD40L in PBMCs as well as in purified populations of T and B cells. Upregulation of CD40L mRNA in PBMCs occurred 3 hours after stimulation with HC and was inhibited by actinomycin D. Upregulation of CD40L mRNA and induction of IgE synthesis by HC were inhibited by the steroid hormone receptor antagonist RU-486. These results indicate that ligand-mediated activation of the glucocorticoid receptor upregulates CD40L expression in human lymphocytes.

Role of JAK3 in CD40-mediated signaling.

CD40 is a member of the tumor necrosis factor receptor family and plays an important role in B-cell survival, growth, differentiation, and isotype switching. Recently, CD40 has been shown to associate with JAK3, a member of the family of Janus Kinases, which are nonreceptor protein kinases involved in intracellular signaling mediated by cytokines and growth factors. To investigate the role of JAK3 in CD40-mediated signaling, we studied the effect of CD40 stimulation on B-cell proliferation, IgE isotype switching, and upregulation of surface expression of CD23, ICAM-1, CD80, and LT-alpha in JAK3-deficient patients. Our studies show that stimulation of B cells with monoclonal antibody to CD40 in the presence of interleukin-4 (IL-4) or IL-13 resulted in similar responses in JAK3-deficient patients and normal controls. This suggests that JAK3 is not essential for CD40-mediated B-cell proliferation, isotype switching, and upregulation of CD23, ICAM-1, CD80, and LT-alpha surface expression.

The superantigen toxic shock syndrome toxin-1 induces CD40 ligand expression and modulates IgE isotype switching.

Isotype switching to IgE requires two signals. The first signal is provided by the cytokines IL-4 or IL-13, and the second signal is delivered by the interaction between the B cell antigen CD40 and its ligand (CD40L) which is expressed on activated T cells. Since superantigens have been shown to activate T cells, we examined the effect of the superantigen toxic shock syndrome toxin-1 (TSST-1) on CD40L expression on T cells. TSST-1 induced expression of CD40L in both freshly isolated T cells and in T cell lines expanded by re-stimulation with TSST-1. CD40L was preferentially expressed in the V beta 2 subset of T cells expanded by TSST-1. We next examined the effect of TSST-1 on IgE synthesis by human peripheral blood mononuclear cells (PBMC). Addition of TSST-1 to PBMC inhibited IL-4-induced IgE synthesis in a dose-dependent manner. This inhibition was reversed partly by adding a neutralizing antibody to IFN-gamma. In contrast, TSST-1 induced high amounts of IgE synthesis in the presence of IL-4 at low T:B cell ratios (0.5:10 to 4:10), a condition which circumvents the inhibitory effect of IFN-gamma. TSST-1 induction of IgE synthesis was inhibited by a mAb to CD40L. These results indicate that superantigens induce CD40L expression in T cells and cause isotype switching in B cells which is mediated by CD40L-CD40 interaction.

Mechanisms of inhibition of IgE synthesis by nedocromil sodium: nedocromil sodium inhibits deletional switch recombination in human B cells.

IgE synthesis requires IL-4 and a T cell-B cell interaction that involves the B-cell antigen CD40 and its ligand expressed on activated T cells. Nedocromil sodium (NS), an effective prophylactic agent in asthma, inhibits IgE synthesis by human B cells. In this report we examined the mechanisms of this inhibition. NS targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in highly purified B cells (greater than 98% CD19+). NS had no effect on the induction of epsilon-germline transcripts by IL-4 but strongly inhibited CD40-mediated S mu --> S epsilon deletional switch recombination. The effect of NS was not specific for CD40 because it inhibited IgE synthesis in B cells stimulated with hydrocortisone plus IL-4. Moreover, the effect of NS was not specific for IgE because it inhibited CD40/IL-4-driven IgG4 synthesis by B cells sorted for lack of surface expression of IgG4. NS caused only modest inhibition of spontaneous IgE synthesis by B cells from patients with hyper-IgE syndrome, suggesting that it has little effect on B cells that have already undergone isotype switching. These results strongly suggest that NS inhibits IgE isotype switching by inhibiting deletional switch recombination and that NS has a novel potential mechanism for the prevention of asthma and other allergic diseases.

Interleukin-4 receptor expression by human B cells: functional analysis with a human interleukin-4 toxin, DAB389IL-4.

BACKGROUND: Studies of human IgE-secreting B cells have proven difficult because of the small size of this population. We have used an interleukin-4 (IL-4) fusion toxin to detect functionally IL-4 receptor (IL-4R) expression on B cells involved in IgE synthesis. METHODS: In diphtheria toxin IL-4 (DAB389IL-4) the receptor-binding domain of diphtheria toxin has been replaced with human IL-4. DAB389IL-4 cytotoxicity depends on IL-4R binding and internalization. RESULTS: Addition of DAB389IL-4 inhibited IgE synthesis induced by IL-4+ anti-CD40 monoclonal antibody or hydrocortisone. IgE inhibition resulted from DAB389IL-4 B-cell cytotoxicity because DAB389IL-4 inhibited IL-4-independent B-cell proliferation. Thus induction of human IgE synthesis involves IL-4R+ cells. In contrast, terminally differentiated, IgE-producing B cells no longer express functional IL-4R because DAB389IL-4 only modestly inhibited ongoing IgE synthesis by B cells from patients with hyper-IgE states and only minimally affected IL-4-induced IgE synthesis in normal B cells when the toxin was added at day 7. Pokeweed mitogen-induced IgM synthesis was sensitive to early but not to late addition of DAB389IL-4. Thus the loss of functional IL-4R immunoglobulin-secreting B cells is independent of isotype switching. CONCLUSIONS: IgE-secreting B cells no longer express functional IL-4R. Therapies for allergic disease that target the IL-4R would not affect IgE-secreting B cells but may block the recruitment of B cells into the IgE-secreting pool. For optimal benefits this approach may be combined with therapies that target IL-4R-, IgE-secreting B cells.

gamma/delta T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes.

T cells expressing gamma/delta T cell receptors home to epithelial tissue and may play a role in immunity to infectious agents and foreign antigens. In an effort to understand the role of gamma/delta T cells in directing B cell responses, we investigated the capacity of human gamma/delta T cells to express CD40 ligand (CD40L) and to drive immunoglobulin (Ig) isotype switching in B cells. A multiple step purification procedure resulted in the recovery of highly pure populations of peripheral blood CD4-CD8- gamma/delta T cells. Neither CD40L surface expression nor CD40L mRNA were detected in unstimulated gamma/delta T cells. Stimulation with phorbol ester and ionomycin induced CD40L mRNA and surface CD40L expression by gamma/delta T cells. Both the percentage of CD40L+ cells and the cell surface density of CD40L were significantly lower in gamma/delta T cells compared to unselected T cells. We further demonstrated that in the presence of neutralizing monoclonal antibody to interferon gamma (IFN-gamma), gamma/delta T cells could induce IgE synthesis in B cells, albeit to a lesser extent than unselected T cells. Furthermore, IgE synthesis driven by gamma/delta T cells was inhibited by monoclonal antibody to CD40L. These observations demonstrate that activated gamma/delta T cells express CD40L and can induce isotype switching in B cells.

Role of protein tyrosine kinases and phosphatases in isotype switching: crosslinking CD45 to CD40 inhibits IgE isotype switching in human B cells.

Protein tyrosine kinases and protein tyrosine phosphatases play an important role in the transduction of signals via antigen receptors in T and B cells, and in CD40-dependent B-cell activation. To examine the role of tyrosine kinases and phosphatases in B-cell isotype switching, we examined the effects of the engagement of the transmembrane phosphatase CD45 on the synthesis of IgE induced by IL-4 and anti-CD40 monoclonal antibody (mAb). Crosslinking CD45 to CD40 using biotinylated mAbs and avidin strongly inhibited CD40-mediated IgE synthesis in IL-4-treated human B cells. CD40/CD45 crosslinking did not affect epsilon germline transcription in B cells stimulated with IL-4, but strongly inhibited induction of S mu/S epsilon switch recombination as detected by a nested primer polymerase chain reaction assay. The B-cell src-type tyrosine kinase lyn, which is activated following CD40 engagement, is a potential target for the effects of CD45 observed in our experiments, because CD45/CD40 crosslinking resulted in the inhibition of CD40-mediated lyn phosphorylation and activation. These results suggest an important role for protein tyrosine kinases and phosphatases in CD40-mediated induction of isotype switching to IgE.

Role of protein tyrosine kinases in CD40/interleukin-4-mediated isotype switching to IgE.

The B-cell antigen CD40 transduces signals, which synergize with interleukin (IL)-4 to induce IgE synthesis in human B cells. IL-4 induces epsilon germline transcription but not mature epsilon transcripts or IgE protein synthesis in B cells. Addition of anti-CD40 monoclonal antibody to IL-4-treated B cells results in deletional S mu--> S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. Because both IL-4 and anti-CD40 induce protein tyrosine phosphorylation in B cells, we investigated the role of protein tyrosine kinase in IL-4/CD40-mediated IgE synthesis. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) or the protein kinase A inhibitor N-2-guanidinoethyl-5-isoquinolinesulfonamide, inhibited IgE synthesis in B cells stimulated with IL-4 and CD40. Genestein and herbimycin, but not H7, inhibited IL-4-driven epsilon germline transcription in B cells. Both genestein and herbimycin, but not H7, inhibited CD40-mediated IgE synthesis in B cells pretreated for 4 days with IL-4 to allow optimal expression of epsilon germline transcripts. Inhibition of IgE synthesis in these cultures was accompanied by inhibition of S mu--> S epsilon deletional switch recombination as assayed by nested polymerase chain reactions. These results suggest that activation of protein tyrosine kinase plays an important role in both the IL-4 and the CD40 signalling pathways that lead to IgE isotype switching in B cells.

Disodium cromoglycate inhibits S mu-->S epsilon deletional switch recombination and IgE synthesis in human B cells.

IgE synthesis requires interleukin 4 (IL-4) and a T-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. IL-4 induces epsilon germline transcription whereas ligation of CD40 results in deletional S mu-->S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. We demonstrate that disodium cromoglycate (DSCG), a drug commonly used for the prophylactic treatment of allergic disease, inhibits T cell-driven IgE synthesis by human B cells at concentrations readily achievable in the course of inhaled therapy for asthma. Inhibition of IgE synthesis by DSCG was not the result of drug toxicity because DSCG did not affect the viability of T and B cells or their proliferation to mitogens. DSCG did not interfere with CD40 ligand expression by T cells but clearly targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in populations of highly purified B cells. DSCG had no effect on the induction of epsilon germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu-->S epsilon deletional switch recombination in IL-4-treated B cells as assayed by nested primer PCR. The effect of DSCG was not specific for CD40-mediated induction of IgE isotype switching because DSCG inhibited IgE synthesis as well as S mu-->S epsilon deletional switch recombination induced by hydrocortisone and IL-4 in B cells. Moreover, the effect of DSCG was not specific for IgE isotype switching because DSCG inhibited the synthesis of IgG4 by B cells sorted for lack of surface expression of IgG4 and stimulated with anti-CD40 and IL-4. DSCG caused only minimal inhibition (< 15%) of spontaneous IgE synthesis by lymphocytes from patients with the hyper-IgE syndrome and did not affect pokeweed mitogen-induced IgG and IgA synthesis by lymphocytes suggesting that it has little effect on B cells that have already undergone isotype switching. These results indicate that DSCG inhibits switching to IgE in B cells and suggest a novel potential mechanism for the prevention of allergic disease by DSCG.

Engagement of CD14 on monocytes inhibits the synthesis of human Igs, including IgE.

Engagement of CD14 on normal human monocytes results in monocyte activation and, furthermore, delivers a negative signal that terminates T cell proliferation. We show herein that engagement of selected CD14 epitopes by specific mAbs inhibited PWM-induced IgM and IgG synthesis, as well as IL-4-dependent IgE synthesis by human PBMCs. Inhibition by anti-CD14 mAb was still evident when the Ab was added after 6 days of culture, suggesting that inhibition targets a late B cell activation event. In experiments that focused on the role of CD14 in IgE regulation, suppression of IgE synthesis after CD14 engagement did not result from the release of inhibitory monokines (IL-10, TGF-beta, and PG). In fact, neutralizing Abs and specific inhibitors did not restore the IgE response and anti-CD14-conditioned monocyte supernatants did not inhibit IgE synthesis. On the other hand, inhibition of IgE synthesis by anti-CD14 mAb was not likely to be caused by the lack of soluble factors with amplificatory effects on the IgE response, because addition of either rIL-6 or rTNF-alpha did not overcome CD14-dependent inhibition of IgE production. IgE inhibition after CD14 engagement was exerted at the B cell level, inasmuch as it was observed not only in T cell-dependent IgE induction i.e., mononuclear cells stimulated with IL-4, but also in T cell-independent systems, i.e., T cell-depleted populations stimulated with IL-4 plus anti-CD40 mAb or IL-4 plus hydrocortisone. These data indicate that CD14 plays a regulatory role in B cell responses.

Sequential switching from mu to epsilon via gamma 4 in human B cells stimulated with IL-4 and hydrocortisone.

The molecular events leading to IgE synthesis in human B cells stimulated with IL-4 and hydrocortisone were analyzed. IL-4, but not hydrocortisone, induced C epsilon germ line transcription. However, hydrocortisone increased the levels of IL-4-induced germ line C epsilon transcripts by twofold and delivered the signal required for transcription of mature C epsilon mRNA. Nested primer polymerase chain reaction of high m.w. DNA revealed deletional switch recombination occurring in B cells sorted for lack of expression of surface IgE and stimulated with both IL-4 and hydrocortisone, but not in B cells stimulated with IL-4 alone or hydrocortisone alone. DNA sequence analysis of 10 switch fragments revealed direct joining of S mu to S epsilon in eight fragments, one of which exhibited an 876-bp deletion in S mu. The ninth fragment contained a 50-bp insertion at the S mu/S epsilon junction, which was likely to be derived from S gamma 4. The sequence of the 10th fragment was consistent with either a 17-bp insertion at the S mu/S epsilon junction derived from S gamma 4 or with a complex 38-bp deletion within S epsilon. Mapping of the switch junction sites showed "hot spots" for recombination within S mu but not within S epsilon. These findings indicate that hydrocortisone induces S mu-S epsilon deletional switch recombination in IL-4-treated B cells, and support a model of sequential isotype switching from IgM to IgE via IgG4.

Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM.

B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease.

Molecular analysis of the induction of immunoglobulin E synthesis in human B cells by interleukin 4 and engagement of CD40 antigen.

The molecular events leading to immunoglobulin E (IgE) synthesis in human sIgE- B cells stimulated with interleukin 4 (IL-4) and anti-CD40 monoclonal antibody (mAb) 626.1 were analyzed. Anti-CD40 mAb increased the levels of IL-4-induced germline C epsilon transcripts and induced the production of mature C epsilon mRNA. These effects were dependent on the presence of IL-4. Nested primer PCR revealed deletional switch recombination occurring only in B cell stimulated with both IL-4 and anti-CD40 mAb. DNA sequence analysis of switch fragments showed direct S mu/S epsilon joining, without the deletions or duplications within S mu often found in B cells stimulated with IL-4 and Epstein-Barr virus. Analysis of the switch junction map sites showed "hot spots" for recombination within S mu, but not within S epsilon. These findings indicate that IL-4 provides a signal to B cells to induce germline C epsilon transcription and concurrent CD40 engagement induces S mu/S epsilon deletional switch recombination, production of mature C epsilon mRNA, and IgE synthesis.

Deletional switch recombination occurs in interleukin-4-induced isotype switching to IgE expression by human B cells.

There is controversy as to whether deletional rearrangement occurs between the IgM and IgE switch regions (S mu and S epsilon, respectively) during switching to the IgE isotype. We have addressed the issue by stimulating normal human B cells, sorted for lack of expression of surface IgE, to produce IgE by infection with Epstein-Barr virus (EBV) in the presence of interleukin 4 (IL-4). Genomic DNA was amplified for S mu/S epsilon switch junction fragments by utilizing the nested-primer polymerase chain reaction. Switch junction fragments were amplified from B cells infected with EBV in the presence of IL-4 but not from B cells infected with EBV alone. The DNA sequence of these "switch fragments" revealed direct joining of S mu to S epsilon in each case. The recombination sites within S mu were clustered within 900 base pairs at the 5' end of the switch region, suggesting that there are "hot spots" for recombination within S mu. The S epsilon recombination sites were scattered throughout the S epsilon region. These findings indicate that IL-4-induced isotype switching to IgE production in human B cells is accompanied by DNA rearrangements with joining of S mu to S epsilon.

Hydrocortisone and IL-4 induce IgE isotype switching in human B cells.

Induction of IgE synthesis in human B cells requires two signals. The first signal is delivered by the cytokine IL-4. The second signal activates B cells and is delivered by T cells, EBV infection, or engagement of the B cell-specific Ag CD40. Hydrocortisone (HC) has recently been shown to synergize with IL-4 to induce IgE synthesis in CD5+ chronic lymphocytic leukemia B cells. We show herein that a combination of HC and rIL-4 induces IgE synthesis in highly purified normal peripheral blood B cells. HC and IL-4 acted directly on B cells, because T cells and monocytes were not required for IgE synthesis. IgE induction was shown to occur in surface IgE- B cells isolated by cell sorting. These results suggest that IgE synthesis results from isotype switching, rather than from expansion of a precommitted B cell population. Furthermore, IgE synthesis was induced in sorted CD5- B cells, indicating that the ability to produce IgE in response to HC and IL-4 is not constrained by CD5 expression. Endogenous IL-6 was critical for induction of IgE synthesis by HC and IL-4, because an anti-IL-6 antibody strongly inhibited IgE production. These data suggest that hormones may play an important role in the regulation of IgE synthesis.

CD40 and IgE: synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells.

A novel pathway of IgE-B cell differentiation has been identified. Engagement of the B cell antigen CD40 by F(ab')2 fragments of monoclonal antibody (mAb) 626.1 in the presence of recombinant interleukin 4 (rIL-4) induced intense IgE synthesis, but modest IgG synthesis, by highly purified human B cells. Surface IgE- B cells isolated by cell sorting were induced to produce IgE by mAb 626.1 and IL-4. Thus, IgE synthesis is unlikely to result from expansion of a B cell population precommitted to IgE in vivo. A neutralizing anti-IL-6 antibody strongly, but not completely, inhibited the IgE response. This indicates that autocrine production of IL-6 plays an important amplification role in IgE synthesis triggered by anti-CD40 mAb and IL-4. Although the exact role played by CD40 in IgE responses in vivo remains to be established, this T cell-independent system represents a useful model to characterize the biochemical and molecular events leading to IgE synthesis in human B cells.

Induction of germ-line and mature C epsilon transcripts in human B cells stimulated with rIL-4 and EBV.

EBV and rIL-4 induce T cell-independent IgE production by normal human B cells. We demonstrate here that EBV and IL-4 induced the synthesis of IgE by surface IgE-negative B cell precursors isolated by cell sorting. This result suggests that the induction of IgE by EBV and IL-4 results not merely from the expansion of a precommitted surface IgE-positive B cell population but more likely from IL-4-directed switching to IgE. At the molecular level, IL-4 and EBV induced the appearance of 2.0- and of 1.8-kilobase (kb) RNA bands, both of which hybridized with an 0.88-kb HinfI fragment spanning part of the C epsilon 1 exon and the entire C epsilon 2 exon. The 1.8-kb band but not the 2.0-kb band also hybridized with a cloned genomic 0.7-kb SmaI fragment located approximately 2 kb upstream of C epsilon. Thus, EBV and IL-4 induced germline (1.8-kb) as well as mature (2.0-kb) C epsilon transcripts. IL-4 by itself induced germ-line C epsilon transcripts but not mature C epsilon transcripts in purified normal B cells. IL-4 failed to induce IgE synthesis in established EBV B cell lines and failed to induce 2.0-kb mature C epsilon transcripts but induced 1.8-kb germ-line C epsilon transcripts. These data show that IL-4 is sufficient for the induction of C epsilon germ-line transcription. In contrast, the transcription of mature epsilon mRNA requires an additional activating signal, provided by infection with EBV. Established EBV transformation results in a dissociation between germ-line C epsilon transcription and the ability to undergo IgE switching in response to IL-4.

Regulation of immunoglobulin (Ig)E synthesis in the hyper-IgE syndrome.

The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis, and recurrent infections. The mechanisms responsible for hyperproduction of IgE in HIE patients are presently unknown. We investigated whether spontaneous in vitro IgE synthesis by PBMC from seven HIE patients was sensitive to signals (cell adhesion, T/B cell cognate interaction and lymphokines: IL-4, IL-6, and IFN-gamma) known to regulate IgE induction in normals. Our results show that, unlike IL-4 dependent IgE synthesis induced in normals, spontaneous IgE production by PBMC from HIE patients was not blocked by monoclonal antibodies to CD2, CD4, CD3, and MHC class II antigens. Furthermore, antibodies to IL-4 and IL-6 did not significantly suppress IgE production. IFN-gamma had no significant effects on spontaneous in vitro IgE synthesis. To test whether an imbalance in lymphokine production might underlie hyperproduction of IgE in HIE patients, mitogen-induced secretion of IL-4 and IFN-gamma by PBMC was assessed. No significant difference was detected between HIE patients and normal controls. Thus, ongoing IgE synthesis in the HIE syndrome is largely independent of cell-cell interactions and endogenous lymphokines, and is due to a terminally differentiated B cell population, no longer sensitive to regulatory signals.