An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.     

The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain

The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.     

Induction of Fc epsilon RII/CD23 on phytohemagglutinin-activated human peripheral blood T lymphocytes. I. Enhancement by IL-2 and IL-4.

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.     

IL-15:IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization

IL-15, a promising cytokine for treating cancer and viral diseases, is presented in trans by the IL-15 receptor (IL-15R) alpha-chain to the IL-15Rβγc complex displayed on the surface of T cells and natural killer (NK) cells. We previously reported that an asparagine to aspartic acid substitution at amino acid 72 (N72D) of IL-15 provides a 4-5-fold increase in biological activity compared to the native molecule. In this report, we describe Chinese hamster ovary (CHO) cell expression of a soluble complex (IL-15 N72D:IL-15RαSu/Fc) consisting of the IL-15 N72D superagonist and a dimeric IL-15Rα sushi domain-IgG1 Fc fusion protein. A simple but readily scalable affinity and ion exchange chromatography method was developed to highly purify the complex having both IL-15 binding sites fully occupied. The immunostimulatory effects of this complex were confirmed using cell proliferation assays. Treatment of mice with a single intravenous dose of IL-15N72D:IL-15RαSu/Fc resulted in a significant increase in CD8+ T cells and NK cells that was not observed following IL-15 treatment. Pharmacokinetic analysis indicated that the complex has a 25-h half-life in mice which is considerably longer than <40-min half-life of IL-15. Thus, the enhanced activity of the IL-15N72D:IL-15RαSu/Fc complex is likely the result of the increased binding activity of IL-15N72D to IL-15Rβγc, optimized cytokine trans-presentation by the IL-15RαSu domain, the dimeric nature of the cytokine domain and its increased in vivo half-life compared to IL-15. These findings indicate that this IL-15 superagonist complex could serve as a superior immunostimulatory therapeutic agent.     

Internalization of interleukin 2 (IL-2) by high affinity IL-2 receptors is required for the growth of IL-2-dependent T cell lines

During the growth of interleukin 2 (IL-2)-dependent T cells IL-2 binding is followed by internalization of the complex between IL-2 and the high affinity IL-2 receptor (HA-IL-2R). The respective role of IL-2 binding to HA-IL-2R and internalization of the complex has been examined. Monoclonal antibody 7D4 (IgM) blocks IL-2-dependent T cell growth although it does not affect IL-2 binding to HA-IL-2R. We show here that 7D4 inhibits T cell growth by blocking IL-2 internalization by HA-IL-2R. In contrast, Fab fragments prepared from 7D4 neither block IL-2 internalization nor inhibit T cell growth. Monoclonal 5A2, that recognizes an epitope related to the IL-2 binding site as well as its Fab fragment, inhibits T cell growth and IL-2 internalization. Monoclonal antibody 7D4, because of its pentameric structure, probably aggregates the IL-2R at the T cell surface and therefore prevents it internalization. The data presented in this paper suggest that simple occupancy of HA-IL-2R by IL-2 is not sufficient to transduce the T cell growth signal; this signal is transmitted only after internalization of the IL-2/HA-IL-2R complex.     

Changing patterns of lymphocyte proliferation, IL-2 production and utilization, and IL-2 receptor expression in mice infected with Schistosoma mansoni

In murine schistosomiasis mansoni the soluble egg Ag (SEA)-induced L3T4+ T cell-mediated circumoval granulomatous response peaks at the acute stage (8w) and undergoes Lyt-1+, Lyt-2+ suppressor cell mediated down-modulation at the chronic stage (20w) of the infection. In the present study lymphoproliferation, IL-2 production, utilization, and IL-2R display were examined in splenic lymphocytes of infected mice. The L3T4+ subset was the major SEA-specific proliferative population at both stages of the infection. Chronic infection spleen cells or the L3T4+ subset proliferated less compared with their acute infection counterparts. Elimination of the Lyt-2+ subset did not improve diminished proliferation. Chronic infection cells and the Lyt-2+ subset stimulated with SEA and exogenous rIL-2 regained their proliferative ability to a level comparable with acute infection cells. Ag-stimulated acute infection T cells produced 40 to 50 chronic infection cells less than 5 U/ml IL-2. Elimination of L3T4+ T cells diminished, and the Lyt-2+ T cells left unchanged IL-2 production in the acute infection cells. Elimination of Lyt-2+ subset from the chronic infection population did not enhance IL-2 production. Exogenously added rIL-2 was equally utilized by acute or chronic infection T cells. Scatchard plots of [125I]IL-2 binding showed similar numbers of high affinity receptors on acute (189) and chronic infection cells (118), but the number of low affinity receptors was higher on the acute (2229) vs the chronic infection lymphocytes (578). Analysis of IL-2R expression by two-color fluorescence and flow cytometry revealed that 30 to 40% of the acute, chronic infection L3T4+ cells displayed receptor. Receptor expression increased by added SEA, or SEA + rIL-2. R display among Lyt-2+ cells was only 12 to 18%. SEA stimulation enhanced receptor display more among the acute, SEA + rIL-2 stimuli raised receptor expression only among chronic infection lymphocytes. These data do not show inherent defect in IL-2R display, or utilization in chronic infection T cells. Diminished IL-2 production appears to be the cause of decreased T cell responsiveness.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

Protein phosphorylation mediated by IL-2/IL-2 receptor beta-chain interaction

We previously found that IL-2 rapidly induced protein phosphorylation of a 67-kDa (pp67) and four 63-kDa (pp63s) cellular proteins in various T cells. Here, we show that the IL-2-stimulated phosphorylation is mediated by the IL-2R beta-chain composed of the high affinity IL-2R, and induced by activation of Ca2+/phospholipid-dependent protein kinase C (PKC). The IL-2-stimulated phosphorylation was always observed in various T cell lines bearing high affinity IL-2R, but never observed in cells which express only low affinity IL-2R consisted of alpha-chain alone. When the expression of high affinity IL-2R was modified by anti-IL-2R mAb for reducing the affinity to 8- to 10-fold lower without affecting the sites of IL-2R, the effective dose of IL-2 on phosphorylation of pp67 increased 8 to 10 times. When cells were treated with pronase, approximately 95% sites of low affinity IL-2R were selectively decreased, but the IL-2 dose dependency for pp67 phosphorylation was little affected. These data exactly suggest that protein phosphorylation in response to IL-2 such as pp67 and pp63s, is mediated by high affinity but not low affinity IL-2R. Furthermore, the IL-2-stimulated phosphorylation of these proteins was also observed in MLA 144 cells which express only low affinity IL-2R consisting of beta-chain alone. In addition, various phorbol esters and tumor promoters, which activate PKC, were also demonstrated to induce the phosphorylation of a pp67 and pp63s in these T cell lines. Therefore, the present study suggests that IL-2/IL-2R beta-chain interaction triggers the phosphorylation of pp67 and pp63s, where the PKC may have an important role.     

Stimulation of the beta-subunit of the IL-2 receptor induces MHC-unrestricted cytotoxicity in T acute lymphoblastic leukemia cells and normal thymocytes

Recently, several laboratories have identified a novel protein(s) of 70,000 to 75,000 Da (IL-2R beta-subunit) that, when expressed with the p55 Tac protein (alpha-subunit), imparts high affinity IL-2 binding. Expression of the beta-subunit mediates acquisition of MHC-unrestricted cytotoxicity in large granular lymphocytes. We report that thymocytes and T acute lymphoblastic leukemia cells express the beta-subunit of the IL-2R in the absence of detectable alpha-subunit expression and that IL-2 induces acquisition of MHC-unrestricted cytotoxic activity in these cells through stimulation of the beta-subunit.     

Selective immunosuppression of activated T cells with the chimeric toxin IL-2-PE40. Inhibition of experimental autoimmune uveoretinitis

A characteristic of activated T lymphocytes is the expression of high affinity IL-2R. We studied a new method of selective immunosuppression directed against activated T cells by using a chimeric recombinant protein (IL-2-PE40) composed of IL-2 fused to a modified Pseudomonas exotoxin lacking its cell recognition domain. As a model of T cell-mediated disease, we used experimental autoimmune uveoretinitis (EAU) produced in Lewis rats by active immunization with the retinal S-Ag. The treatment protocol consisted of i.p. injection of IL-2-PE40 at 0.25 micrograms/g every 12 h. Controls were PBS, PE40, or IL-2-PE40asp553 a mutant form of the molecule with reduced activity. Treatment with IL-2-PE40 resulted in a significant reduction of the incidence and severity of EAU over controls. The analysis of the effect of i.p. injection of IL-2-PE40 on the popliteal draining lymph nodes of immunized animals showed a marked reduction in the lymphocytes content. Transfer experiments demonstrated that IL-2-PE40 prevented the development of EAU effector T cells. Interestingly, although activated B cells were reported to express IL-2R, there was no significant reduction of antibody production against the immunizing Ag under IL-2-PE40 treatment, suggesting sparing of the B cells.     

The role of the IL-2 pathway in costimulation blockade-resistant rejection of allografts.

Blockade of the CD40 and CD28 costimulatory pathways significantly prolongs allograft survival; however, certain strains of mice (i.e., C57BL/6) are relatively resistant to the effects of combined CD40/CD28 blockade. We have previously shown that the costimulation blockade-resistant phenotype can be attributed to a subset of CD8+ T cells and is independent of CD4+ T cell-mediated help. Here we explore the role of the IL-2 pathway in this process using mAbs against the high affinity IL-2R, CD25, and IL-2 in prolonging skin allograft survival in mice receiving combined CD40/CD28 blockade. We have also investigated the effects of treatment on effector function by assessment of cytotoxicity and the generation of IFN-gamma-producing cells in response to allogeneic stimulators as well as proliferation in an in vivo graft-vs-host disease model. We find that additional blockade of either CD25 or IL-2 significantly extends allograft survival beyond that in mice receiving costimulation blockade alone. This correlates with diminished frequencies of IFN-gamma-producing allospecific T cells and reduced CTL activity. Anti-CD25 therapy also synergizes with CD40/CD28 blockade in suppressing proliferative responses. Interestingly, depletion of CD4+ T cells, but not CD8+ cells, prevents prolongation in allograft survival, suggesting an IL-2-independent role for regulation in extended survival.     

A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.     

IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.     

TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.     

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.     

Purified Fc epsilon R+ bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin.

Non-B, non-T cells from spleen and bone marrow cells produce IL-4 in response to cross-linkage of high affinity receptors for Fc epsilon R or Fc gamma RII, and to treatment with calcium ionophores. Cells bearing high affinity Fc epsilon R constituted 1 to 2% of non-B, non-T cells of spleen and of total bone marrow cells from naive donors. In mice whose immune systems had been polyclonally activated by injection with anti-IgD antibodies or had been infected with Nippostrongylus brasiliensis larvae, the frequency of Fc epsilon R+ cells in splenic non-B, non-T cells was also 1 to 2% but in bone marrow from anti-IgD-injected mice donors the frequency was approximately 5%. Cell sorting experiments revealed that all of the capacity to produce IL-4 in response to immobilized IgE or IgG2a or to ionomycin was found in the Fc epsilon R+ fraction. Among the Fc epsilon R+ spleen cells from naive donors, the frequency of IL-4-producing cells was 1/20 to 1/40 whereas in mice that had been injected with anti-IgD or infected with N. brasiliensis, the frequency of IL-4 producing cells in the Fc epsilon R+ population was approximately 1/5.