Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM

In this report, we describe a novel biologic activity of IL-4 namely, its ability to induce activated human B cells to produce IgM. Staphylococcus aureus Cowan I-activated blasts prepared from high density tonsil B cells were found to secrete IgG and IgM, but no IgE, when cultured in the presence of rIL-4. The differentiating activity of rIL-4 was totally blocked by a neutralizing anti-IL-4 antiserum, therefore demonstrating that the IgG/IgM-inducing activity of rIL-4 was an intrinsic property of IL-4. rIL-4 was only minimally inducing Ig production of blasts prepared from low density B cells, whereas it induced B cell blasts prepared from high density B cells to secrete a high amount of Ig. Delayed additions of the neutralizing anti-IL-4 antiserum demonstrated that a 48-h contact between IL-4 and B cell blasts was required for optimal Ig production. The IL-4-mediated IgG and IgM production was neither suppressed by IFN-gamma nor by anti-CD23 mAb 25, whereas these agents have been shown earlier to inhibit IgE production of enriched B cells cultured in the presence of IL-4. These data indicate that the IgG/IgM-inducing activity of IL-4 is not regulated like the IL-4-induced IgE production by enriched B cells.     

IgE production by normal human B cells induced by alloreactive T cell clones is mediated by IL-4 and suppressed by IFN-gamma

Seven T cell clones were established from mixed leukocyte cultures in which PBMC from two healthy donors and from one patient suffering from the hyper-IgE syndrome were stimulated by the irradiated EBV-transformed B cell lines JY or UD53. Five of seven T cell clones, after activation by co-cultivation with JY or UD53 cells, induced a low degree of IgE production by normal blood B cells. In one experiment in which the normal B cells could activate the T cell clones directly, IgE production was also observed in the absence of the specific stimulator cells. IgE production was also obtained with supernatants of the T cell clones collected 4 to 5 days after activation by their specific stimulator cells. In addition, the supernatants induced IgG, IgA, and IgM synthesis. All seven clones produced variable concentrations of IL-4 and IFN-gamma. The clones FA-28 and BG-39, which failed to induce IgE synthesis, produced, compared with the other clones tested, relatively high quantities of IFN-gamma (4700 and 2500 pg/ml, respectively). These high levels of IFN-gamma accounted for the lack of induction of IgE synthesis, because in the presence of a polyclonal anti-IFN-gamma antiserum, supernatants of FA-10 and BG-39 induced significant IgE production. In addition, the low degree of IgE production induced by supernatants of two other T cell clones (FA28 and BG24) was 15- and 3-fold enhanced, respectively, in the presence of the anti-IFN-gamma antiserum. IgE synthesis by normal B cells was also induced by rIL-4, indicating that IL-4 present in T cell clone supernatants was responsible for induction of IgE production. This notion was supported by the finding that IgE production induced by supernatant of BG-24 was strongly inhibited by a polyclonal anti-IL-4 antiserum. In contrast, IgG and IgA production induced by supernatant of BG-24 were not significantly affected by the anti-IL-4 antiserum. Only a slight inhibition of IgM synthesis was observed. Collectively, our results indicate that both recombinant and naturally produced IL-4 induce normal human B cells to synthesize IgE. However, final IgE production induced by T cell clone supernatants is the net result of the inducing and suppressive effects of IL-4 and IFN-gamma respectively, that are secreted simultaneously by the T cell clones upon activation.     

Human atopen-specific types 1 and 2 T helper cell clones.

Eight representative T lymphocyte clones (TLC) randomly selected from previously described panels of CD4+ housedust mite Dermatophagoides pteronyssinus (Dp)-specific TLC from atopic and nonatopic donors were studied in more detail in a comparative investigation. The TLC from the atopic donors closely resembled murine type 2 Th (Th2) cells by secreting substantial IL-4, IL-5, IL-6, TNF-alpha, and granulocyte-macrophage (GM)-CSF, minimal IFN-gamma, and relatively little IL-2. In contrast, the nonatopic's TLC resembled murine type 1 Th (TH1) cells by secreting substantial IFN-gamma, IL-2, TNF-alpha, and GM-CSF, no IL-4, and little IL-5. A difference with murine Th1 cells was their additional secretion of IL-6. These cytokine profiles were consistent upon stimulation via different activation pathways including stimulation with specific Dp Ag, mitogenic lectins, and antibodies to CD2, CD3, or CD28. The observed differences in IL-2 secretion, however, were most evident upon stimulation with anti-CD28. If TLC cells were cultured with highly purified B cells and stimulated with anti-CD3 in the absence of exogenous IL-4, IgE synthesis was induced only in cultures with the atopics' Th2 clones, which could be completely abrogated by anti-IL-4. The mere presence of exogenous rIL-4, however, did not result in IgE synthesis, nor did unstimulated TLC cells alone. But if unstimulated TLC cells (that proved not to secrete detectable amounts of cytokines) were added together with rIL-4, again IgE synthesis was induced only in cultures with the atopics' Th2 clones, suggesting the involvement of an additional, as yet unidentified accessory helper function of the atopics' Th2 clones for IgE induction. Unstimulated Th2 clones showed a significantly higher expression of CD28 than the Th1 clones, but three days after stimulation, CD28 expression was elevated to comparable levels on both subsets. When added to B cells at this time point, together with rIL-4 and anti-IFN-gamma, still only the atopics' Th2 clones supported IgE synthesis, arguing against a role for CD28 in this accessory helper function. Whereas the atopics' Th2 clones were excellent helper cells for IgE induction, a unique property of the nonatopic's Th1 clones was their cytolytic activity toward autologous APC which could be induced by specific Dp Ag and by anti-CD3. The present data provide clear evidence for the existence of Th1 and Th2 cells in man.     

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.     

Kinetics of interleukin-4 induction and interferon-gamma inhibition of IgE secretion by Epstein-Barr virus-infected human peripheral blood B cells.

Interleukin-4 (IL-4) acts directly on purified human peripheral blood B cells cultured in the presence of Epstein-Barr virus (EBV) to induce IgE secretion and to enhance the secretion of IgG and IgM. Interferon-gamma (IFN-gamma) inhibits IgE secretion in this system, without affecting the secretion of the other Ig isotypes. To identify the time period during which EBV-infected B cells can be induced by IL-4 to secrete IgE, we have studied the effects of delayed addition of IL-4, or the termination of IL-4 stimulation by wash out or by neutralization with anti-IL-4 antibodies, on the induction of an IgE response. To induce a maximal IgE response, IL-4 had to be added to cultures of B cells plus EBV no later than 2 days after the initiation of culture, and had to remain present through the tenth day of culture. These two time points correspond to the initiation of detectable DNA synthesis (Days 3 to 4) and the earliest detectable Ig secretion (Days 10 to 12) by EBV-stimulated B cells. No IgE response was induced if the period during which EBV-stimulated B cells were cultured with IL-4 was less than 4 days, or if IL-4 were added later than the tenth day of culture, regardless of how long the culture was continued beyond that time. In contrast, IL-4 considerably enhanced IgG and IgM secretion and B cell CD23 expression, even if it was added after the tenth day of culture. IFN-gamma strongly inhibited the IgE response of B cells cultured with IL-4 plus EBV if added within 6 days of the initiation of culture, but had little effect on the generation of IgM or IgG responses made by these cells, regardless of the time of addition. Neither IL-4 nor IFN-gamma affected ongoing IgE secretion by an established, IgE-secreting, EBV-transformed cell line. These observations suggest that: (i) IL-4 first becomes able to induce EBV-activated B cells to secrete IgE as these cells begin to synthesize DNA, must stimulate B cells for at least 4 days to induce IgE secretion, and loses its ability to induce IgE secretion as these cells differentiate into Ig-secreting cells; (ii) the ability of IFN-gamma to suppress an IgE response is limited to this same time period; and (iii) IL-4 enhancement of CD23 expression and IgM and IgG secretion are independent of IL-4 induction of an IgE response.     

Noncognate contact-dependent B cell activation can promote IL-4-dependent in vitro human IgE synthesis

We have previously shown that IL-4 is an essential mediator for the synthesis of human IgE in vitro. In this study we demonstrate that prior physical contact with T cells is required by B cells to synthesize IgE in response to IL-4. Both autologous and allogeneic freshly prepared T cells were consistently able to support IL-4-dependent IgE synthesis, provided that they were added to B cells together with, or before, the addition of IL-4. In addition, most CD4+, as well as a proportion of CD8+, PHA-induced T cell clones (TCC) established from two HLA-DR incompatible donors, supported, in the presence of exogenous IL-4, the synthesis of IgE in B cells from the majority of individuals tested including both donors of cloned T cells. An alloreactive TCC able to produce IL-4 in response to HLA-DR4+ B cells and to induce HLA-DR4+ B cells to synthesize IgE, acquired the ability to support IgE synthesis by B cells lacking the appropriate alloantigen provided that exogenous IL-4 was added. Although the ability of freshly prepared T cells to support IgE synthesis was consistently abrogated by fixation with paraformaldehyde (PF), such a treatment variably affected the IgE-inducing ability of TCC. Preactivation with anti-CD3 before treatment with PF maintained or even enhanced the ability of TCC to support IL-4-dependent IgE synthesis. More importantly, preactivation with anti-CD3, followed by fixation with PF, enabled TCC, apparently devoid of IgE-inducing activity in unfixed condition, to support IL-4-dependent IgE synthesis. Taken together these data suggest that at least two signals are involved in the triggering of human B cells to IgE production: the first is delivered by a T-B cell contact and the second by IL-4. The physical signal delivered by T cells does not necessarily consist of cognate interaction. Non-cognate contact-dependent induction of B cells to IgE synthesis in response to IL-4 appears to be related to molecule(s) distinct from the TCR/CD3 complex, but fully expressed on the membrane of TCR/CD3-activated T cells.     

IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21R gene

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.     

Modulation of IL-4-induced human IgE production in vitro by IFN-gamma and IL-5: the role of soluble CD23 (s-CD23)

IL-4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL-4-induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN-gamma, IFN-alpha, and prostaglandin E-2 (PGE-2). These substances also inhibited IL-4-induced CD23 expression and subsequent release of soluble CD23 (s-CD23). In addition, IgE production was blocked by F(ab')2 fragments of an mAb against CD23. In contrast, IL-5 enhanced IL-4-induced IgE production, provided IL-4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s-CD23. Collectively, these results indicate that there is a correlation between s-CD23 release and IgE production. However, s-CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI-8866 was ineffective in inducing IgE production in the absence of IL-4, but acted synergistically with suboptimal concentrations of IL-4. In addition, it is demonstrated that alloreactive T-cell clones produced varying concentrations of IL-4, IL-2, or IFN-gamma upon stimulation. Only supernatants of 2/4 of these T-cell clones induced a low degree of IgE synthesis, but in the presence of anti-IFN-gamma antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti-IL-4 antibodies, indicating that IL-4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL-4-induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL-5 and s-CD23 but down-regulated by IFN-gamma, IFN-alpha, and PGE-2, respectively.     

Inability of IL-12 to down-regulate IgE synthesis due to defective production of IFN-gamma in atopic NC/Nga mice.

NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-gamma production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-gamma to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-gamma by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-gamma may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.     

IL-4 requirements for the generation of secondary in vivo IgE responses.

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.     

Superinduction of the murine B cell Fc epsilon RII by T helper cell clones. Role of IL-4

Th cell clones are known to induce an IL-4 dependent polyclonal IgE synthesis. Because IL-4 can induce the expression of the low affinity FcR for IgE (Fc epsilon RII) the ability of Th cell clones to induce Fc epsilon RII on purified splenic B cells was analyzed. It was found that a TH2 clone could cause a 50- to 100-fold superinduction of Fc epsilon RII after 2 days in culture; after 3 days, the Fc epsilon RII levels had almost returned to base line. The superinduction was inhibited by an anti-IL-4 antibody, 11B11, indicating its dependence on IL-4. A TH1 clone could cause a modest (four fold) induction of Fc epsilon RII, and this induction was not influenced by 11B11. A similar Fc epsilon RII induction was seen when using the supernatant from activated TH1 cells. The component(s) causing this relatively low level Fc epsilon RII induction is not known; a variety of known lymphokines were tested, and only IL-4 demonstrated any capacity for Fc epsilon RII induction on LPS-activated B cells. Addition of rIL-4 at concentrations of 400 U/ml or greater to the TH1 culture was sufficient to cause a Fc epsilon RII superinduction similar to that seen with the TH2 clone, while 40 U/ml was not. In order to determine a potential role for the Fc epsilon RII or its soluble fragment on the IgE synthesis mediated by TH2, a monoclonal anti-Fc epsilon RII, B3B4, was added to the culture. The addition of B3B4 did not have an influence on IgE levels in this system.     

T regulatory-1 cells induce IgG4 production by B cells: role of IL-10

The study was aimed to find out whether T cells with a regulatory profile could regulate the secretion of IgG4. Using tetanus Ag we found that PBMC of healthy human donors responded to exogenous IL-10 by down-regulating IgG1 and increasing IgG4 secretion. IgE was not affected. To investigate the direct effect of IL-10-producing T cells on B cells, we generated T cell clones (TCC) with two different cytokine profiles: first, IL-10high, IL-2low, IL-4low TCC, and second, IL-10low, IL-2high, IL-4high. The T cell-dependent Ab secretion was measured by coculturing purified CD19+ B cells and the TCC. Interestingly, we found that IgG4 production in the coculture correlated with the TCC production of IL-10 (r2 = 0.352, p = 0.0001), but not with IL-2, IL-4, nor IFN-gamma. IgE showed only a trend with regard to IL-4. Further, there was decreased Ab secretion in the absence of T-B cell contact. IL-10 also induced IgG4 when added to a Th1 TCC-B cell coculture system. The present study thus shows that in T-B cell coculture, IL-10, if induced by the TCC or added to the system, down-regulates the immune response by inducing IgG4 secretion. This establishes a direct implication of IL-10 in humoral hyporesponsiveness, particularly in compartments where the T-B cell interplay determines the subsequent immune response. The correlation between IgG4 and IL-10 (r2 = 0.352) indicates that IL-10 is an important but not the only factor for IgG4 induction.     

Participation of IL-4 in the formation of IgD-binding factors by antigen-primed mouse spleen cells

BALB/c mouse spleen cells primed with either keyhole limpet hemocyanin or DNP-keyhole limpet hemocyanin formed not only IgG-binding factors (BF) and IgE-BF but also IgD-BF upon antigenic stimulation. Analysis of cellular mechanisms involved in the formation of Ig-BF by antigenic stimulation revealed that Ag-primed Th cells released lymphokines upon antigenic stimulation, and that the lymphokine(s) in turn stimulates unprimed T cells to form Ig-BF. Normal unprimed lymphocytes formed IgD-BF upon incubation with culture supernatants of Ag-stimulated spleen cells. The formation of IgD-BF induced by the culture supernatant was prevented by anti-IL-4 mAb (11B11). It was also found that 0.3 to 10 U/ml mouse rIL-4, but none of the rIL-1, IL-2, and IFN-gamma, induced normal T cells to form IgD-BF. Indeed, both IL-2 and IFN-gamma inhibited IL-4 to induce the formation of IgD-BF. In contrast, 10 to 50 U/ml of IFN-gamma induced the formation of IgE-BF, and 50 to 200 U/ml IFN-gamma induced the formation of IgG-BF. However, none of the other lymphokines tested, i.e., IL-1, IL-2, and IL-4, induced the formation of either IgE-BF or IgG-BF. The IgD-BF formed by antigenic stimulation of keyhole limpet hemocyanin-primed spleen cells and those formed by stimulation of normal lymphocytes with 1 to 2 U/ml IL-4 enhanced both IgM and IgG1 plaque-forming cell responses of SRBC-primed spleen cells to homologous Ag. In contrast, 1 to 2 U/ml of IL-4, which could induce the formation of IgD-BF, failed to affect the PFC responses. It appears that the formation of IgD-BF may be involved in the effects of IL-4 to enhance the antibody response.     

IL-12 and IL-18 are increased and stimulate IFN-gamma production in sarcoid lungs

Sarcoidosis is a systemic chronic granulomatous disease of unknown cause. Recent investigations revealed that the cytokine profile in inflamed lesions of sarcoidosis is Th1 dominant. To obtain better immunopathologic understanding of sarcoidosis, we examined the expression of IL-12 and IL-18 and their roles in IFN-gamma production in pulmonary sarcoidosis. Sarcoid cases had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in bronchoalveolar lavage (BAL) fluids compared with healthy subjects. IL-12 p70 and IL-18 were immunohistochemically expressed in the epithelioid cells and giant cells of sarcoid granulomas. Significant induction of IFN-gamma, IL-12 p70, and IL-18 was observed from sarcoid BAL fluid cells with LPS stimulation, whereas LPS tended to induce only IL-12 p70 in BAL fluid cells from healthy subjects. Sarcoid cases had significantly greater IFN-gamma induction with LPS stimulation than healthy subjects did. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells as well as in LPS-stimulated sarcoid BAL fluid cells, but IFN-gamma and IL-12 mRNA expression was observed only in LPS-stimulated BAL fluid cells. Treatment with anti-IL-12- and anti-IL-18-neutralizing Abs significantly inhibited IFN-gamma production from LPS-stimulated BAL fluid cells of sarcoid cases. Coadministration of rIL-12 or rIL-18 induced greater IFN-gamma production in sarcoid BAL fluid cells than in normal BAL fluid cells. We concluded that bioactive IL-12 and IL-18 were produced in sarcoid BAL fluid cells and synergistically induced IFN-gamma production, indicating important cytokines in the Th1 response of sarcoidosis.     

Influence of recombinant IL-4, IFN-alpha, and IFN-gamma on the production of human IgE-binding factor (soluble CD23)

This study documents the influence of rIL-4, IFN-gamma, and IFN-alpha on the production of IgE-BF and the expression of lymphocyte receptor for IgE or CD23 Ag (Fc epsilon R II) by human mononuclear cells. IL-4 increases the secretion of IgE-binding factor (BF) by highly purified B lymphocytes, adherent cells, and U937 monoblastic cells. The effect of IL-4 on purified B cells is augmented by costimulating the cells with F(ab')2 anti-IgM. IFN-gamma, IL-2, IL-1-alpha, or IL-1 beta and the low m.w. B cell growth factor have no effect on IgE-BF production by purified B cells even when they are used in combination with anti-IgM. Stimulation of purified T cells with IL-4 or IL-4 plus PMA leads to the production of very small amounts of IgE-BF that might well be derived from the contaminating non-T cells. IFN-gamma increases IgE-BF synthesis by unfractionated PBMC, T cell-depleted PBMC, adherent cells, and U937 cells suggesting that it induces monocytes to release IgE-BF, IFN-gamma suppresses the IL-4-induced Fc epsilon R II expression and IgE-BF production by highly purified B cells but not by PBMC or their T cell-depleted fractions. IFN-alpha inhibits IgE-BF production by IFN-gamma-stimulated PBMC and by IL-4-stimulated cells suggesting that it exerts its effect on B cells and on monocytes. Moreover IFN-alpha suppresses the IL-4-induced expression of Fc epsilon R II on B cells. Both IFN-alpha and IFN-gamma suppress the synthesis of IgE by PBMC in response to IL-4. Taken collectively the results indicate that: 1) IL-4 induces IgE-BF production by both B cells and monocytes, 2) IFN-gamma stimulates IgE-BF synthesis by monocytes but suppresses its production by IL-4-stimulated B cells, and finally 3) IFN-alpha inhibits IgE-BF synthesis in response to either IFN-gamma or IL-4.     

Generation of B cell memory and affinity maturation. Induction with Th1 and Th2 T cell clones.

We used an adoptive transfer system and CD4+ T cell clones with defined lymphokine profiles to examine the role of CD4+ T cells and the types of lymphokines involved in the development of B cell memory and affinity maturation. Keyhole limpet hemocyanin (KLH)-specific CD4+ Th2 clones (which produce IL-4 and IL-5 but not IL-2 or IFN-gamma) were capable of inducing B cell memory and affinity maturation, after transfer into nude mice or after transfer with unprimed B cells into irradiated recipients and immunization with TNP-KLH. In addition, KLH-specific Th1 clones, which produce IL-2 and IFN-gamma but not IL-4 or IL-5, were also effective in inducing B cell memory and high affinity anti-TNP-specific antibody. The induction of affinity maturation by Th1 clones occurred in the absence of IL-4, as anti-IL-4 mAb had no effect on the affinity of the response whereas anti-IFN-gamma mAb completely blocked the response. Th1 clones induced predominantly IgG2a and IgG3 antibody, although Th2 clones induced predominantly IgG1 and IgE antibody. We thus demonstrated that some Th1 as well as some Th2 clones can function in vivo to induce Ig synthesis. These results also suggest that a single type of T cell with a restricted lymphokine profile can induce both the terminal differentiation of B cells into antibody secreting cells as well as induce B cell memory and affinity maturation. Moreover, these results suggest that B cell memory and affinity maturation can occur either in the presence of Th2 clones secreting IL-4 but not IFN-gamma, or alternatively in the presence of Th1 clones secreting IFN-gamma but not IL-4.     

IL-4-based helper activity of CD4 T cells is radiation sensitive

The capacity of CD4⁺ T cells to induce IgG synthesis in B cells has been known to be radioresistant for more than 20 years. However, the radiation sensitivity of helper T cells with regard to their ability to induce the synthesis of isotypes other than IgG has not been studied. We therefore irradiated KLH-primed lymph node T cells and examined their capacity to induce IgG, IgM, and IgE synthesis in hapten-primed B cells. We demonstrated that while the capacity of KLH-primed lymph node cells to induce IgG synthesis was not affected by irradiation, the capacity of such T cells to induce IgE synthesis was greatly reduced by γ-irradiation. This was consistent with our observations that IL-4 and IL-5 synthesis in such cells was greatly diminished by irradiation, whereas IL-2 synthesis was only minimally affected. A similar differential sensitivity to irradiation of the helper activity of Th1 and Th2 clones was observed with regard to their ability to induce IgE and IgG synthesis under cognate conditions. Irradiation greatly inhibited the capacity of Th2 clones to induce IgE synthesis, but only minimally affected the capacity of Th1 clones to induce IgG synthesis in primed B cells. The capacity of irradiated Th2 clones to induce IgE synthesis was restored by the addition of IL-4 and IL-5. These results taken together indicated that the sensitivity to irradiation of T helper cells with regard to the induction of IgE but not IgG synthesis was due to the sensitivity to irradiation of the production of IL-4 but not of IL-2. Thus, although some functions of CD4⁺ T cells are resistant to radiation, other functions, particularly those that depend on the production of IL-4 and IL-5, are greatly diminished by ionizing radiation.