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

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

The action of interleukin-4 on antigen-specific IgG1 and IgE production by interaction of in vivo primed B cells and carrier-specific cloned Th2 cells

The production of anti-trinitrophenyl (TNP) antibodies of different isotypes from in vivo primed B cells was studied using the plaque-forming cell method. It was shown that these B cells secrete anti-trinitrophenyl antibodies of different isotypes only in the presence of Th2 cells specific for keyhole limpet hemocyanin (KLH) and the hapten-carrier conjugate TNP-KLH. Lipopolysaccharide-stimulated primed B cells without cells from the Th2 clone did not produce anti-TNP-specific IgG1 or IgE antibodies even in the presence of the hapten-carrier antigen TNP-KLH. Supernatants from these Th2 clones cultured with antigen-presenting cells and the complete antigen were unable to activate primed B cells for antibody secretion. Cognate interaction between primed B cells and carrier-specific Th2 cells is a prerequisite for hapten-specific IgG1 or IgE production. Anti-IL-4 antibody inhibited secretion of anti-hapten IgE antibody. Therefore, for production of anti-hapten antibody of the IgE isotype IL-4 is also necessary.     

Nonspecific enhancement of mouse antihapten IgE antibody response: Involvement of a T-cell subpopulation and its product for the potentiation

A concomitant administration of Nippostrongylus brasiliensis saline extract (Nb) with dinitrophenylated ovalbumin (DNP-Ov) significantly enhanced the anti-DNP IgE antibody response in mice which had been irradiated and given a combination of spleen and mesenteric lymph node cells from syngeneic, infected donors and cells from DNP-keyhole limpet hemocyanin (DNP-KLH)-primed donors. The treatment of lymphocytes from infected mice with anti-mouse brain-associated θ serum and C abrogated the enhancing activity. The potentiation occurred in mice receiving nylon wool-nonadherent cells but not in mice receiving adherent cells. Challenge with Nb plus DNP-Ov failed to induce potentiation in C3H mice which are known as nonresponders to low doses of Ov, whereas challenge with Nb plus DNP-bovine gamma globulin (BGG) potentiated the response. However, further increase of the enhanced response was not obtained by adding carrier (BGG or Ov)-primed cells to the transferred lymphocyte populations. When a T-independent antigen, DNP-Ficoll, was used for challenge concomitantly with antigen Nb, no potentiation occurred, even though DNP-Ficoll did not give any tolerogenic or suppressive effect on the IgE antibody response to DNP-Nb. An enhancing activity on the IgE class of antibody response but not on the IgG class was observed in supernatants of in vitro culture of lymphocytes from infected mice upon stimulation of the cells with 10 to 50 μg Nb. These results indicate that the potentiation is mediated by Nb specific T cells via a soluble factor(s) that enhances specifically the IgE class of antibody responses but nonspecifically in terms of antigens used for immunization. The results also suggest that the potentiating factor displays its activity in the presence of other T cells reactive to carrier determinants of the challenging antigen but not of cells which already have committed themselves to the carrier and differentiated as helper cells.     

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.     

IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.     

Relationships between cell-mediated immunity and the IgE antibody response: II. Delayed hypersensitivity and antibody production to DNP-Ascaris conjugates

Rats of the W/F strain were immunized with DNP-Ascaris conjugates using complete Freund's adjuvant (CFA), Al(OH)₃ gel (alum), or B. pertussis vaccine as adjuvants. Cell-mediated immunity was assessed by lymphotoxin in vitro and by delayed hypersensitivity in vivo. IgE and IgG antibody determinations were made on serum pools obtained at various times during the primary and secondary responses. Although delayed hypersensitivity appeared earlier than lymphotoxin, these two parameters correlated during the primary but not during the secondary response. The discrepancies suggested that different cells may be responsible for these two phenomena. Antibody production was influenced by the adjuvant used. CFA led to IgG antibody responses to both hapten and carrier but not to IgE antibody production. The use of B. pertussis resulted in both IgE and IgG antibody production. In the case of alum, anti-hapten antibodies appeared during the primary response while anti-carrier antibodies of both IgE and IgG classes were detected after booster. The results indicated that cell-mediated immunity, IgE, and IgG antibodies appeared independently in an ordered, temporal sequence, and that these responses were not mutually exclusive but were under strong modulatory influences of the various adjuvants used.     

Humoral immune functions in IL-4 transgenic mice

We have analyzed mice expressing IL-4 as a transgene, and found that expression of this lymphokine has profound effects on B cell function. B cells from transgenic mice exhibit phenotypic changes, including an increase in size and elevated expression of class II MHC. IL-4 increases the quantity of IgE produced by transgenic-derived B cells in response to LPS stimulation. In vivo, IL-4 markedly affects the serum Ig isotype repertoire. Serum levels of IgG1 and IgE are elevated, and levels of IgG2a, IgG2b, and IgG3 are depressed in IL-4 transgenic mice. Ag-specific antibody responses to immunization with hapten-carrier conjugates are also affected by IL-4. Transgenic mice show increased anti-hapten IgE and IgG1 and reduced anti-hapten IgG2a, IgG2b, and IgG3, compared with wild-type mice. Ag-specific IgE is substantially induced by T cell-dependent Ag, but not T cell-independent Ag, suggesting that cognate T-B interactions in addition to IL-4 are required for generating IgE responses in vivo. In vivo treatment with the anti-IL-4 mAb 11B11 reverses many of the isotype alterations in the transgenic mice, indicating that these changes arise as a direct consequence of IL-4 secretion.     

Induction and properties of DNP-specific T cells in mice sensitized to DNP-coupled mycobacterium.

Splenic T lymphocytes from mice sensitized to 100 microgram of DNP-coupled mycobacterium (DNP-Tbc) showed in vitro proliferative response against DNP- or TNP-conjugated protein antigens. The increased uptake of 3H-thymidine induced by DNP-HSA was partially inhibited by the addition of 10(-4)M DNP-EACA. DNP-AECM-Ficoll did not induce any significant proliferative responses in DNP-Tbc-primed T cell population. However, priming with DNP-Tbc augmented anti-DNP IgG antibody response induced with DNP-Ficoll. The augmentation of IgG response was not due to the presence of DNP-primed B cells or anti-DNP antibody. The results showed that the priming with DNP-Tbc induced DNP-reactive T helper cells which could be triggered with DNP-Ficoll. The possible role of mycobacterium in the induction of hapten-specific T helper cells is discussed.     

Effect of Nippostrongylus brasiliensis infection on anti-hapten IgE antibody response in the mouse: I. Induction of carrier specific helper cells

Inoculation of infective larvae of Nippostrongylus brasiliensis into A/J, BALB/c, and SJL mice primed intraperitoneally (ip) 3 weeks before infection with 1 μg of dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) mixed with 1 mg Al(OH)₃ induced a carrier effect on anti-DNP IgE and IgG₁ antibody responses when the experimental mice were secondarily immunized with an ip injection of 1 μg of DNP-coupled N. brasiliensis extract (DNP-Nb) plus alum 2 weeks after infection. The magnitude of the hapten specific antibody response did not correlate rigidly with the number of larvae in the inoculum. Thus, a dose of 100 larvae was as effective in inducing the carrier effect as a dose of 800 larvae. Kinetic studies in A/J and BALB/c mice revealed that the anti-DNP IgE antibody response reached a maximum titer 7 days after the secondary immunization. These studies also showed that the enhanced IgE antibody response persisted for more than 40 days, while the response in all control groups terminated prior to that time. Using the adoptive transfer system, it was demonstrated that lymphoid cells obtained from the spleens or the mesenteric lymph nodes of infected mice cooperated with DNP-KLH primed cells to produce hapten specific IgE and IgG, antibodies when the challenge was made with DNP-Nb but not when it was made with 1 μg DNP-ovalbumin, clearly indicating carrier specificity. The helper activity of the cells obtained from infected mice was completely abolished or greatly reduced by the in vitro treatment with anti-θ serum and complement. The helper cells with maximum activity were present as early as 14 days after inoculation. The level of helper activity gradually decreased after 14 days. The results indicate that N. brasiliensis infection is effective in inducing carrier specific helper cells of thymic origin (T cells) in anti-DNP antibody responses. These results confirm those obtained by other investigators and add the new observation that N. brasiliensis infection elicits special helper T cells which induce an enhancement as well as a prolongation of anti-DNP IgE antibody response.     

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

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

Polyclonal activation of the murine immune system by a goat antibody to mouse IgD. IX. Induction of a polyclonal IgE response

The possibility that injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) that stimulates polyclonal IgG1 secretion might also stimulate differentiation of B cells into IgE-secreting cells was suggested by the observation that such treatment induces T cells from those mice to secrete a lymphokine, B cell stimulatory factor 1 (BSF-1), that can stimulate both IgG1 and IgE secretion in vitro. Studies described in this paper show that injection of BALB/c mice with 200 to 3200 micrograms of GaM delta greatly increased the quantity of splenic epsilon chain-encoding mRNA, the number of spleen cells with cytoplasmic IgE, and the concentration of serum IgE 7 days after injection. Serum IgE levels obtained in these mice were approximately 100 times baseline levels and were comparable with those found in mice infected with the nematode parasite Nippostrongylus brasiliensis, but were approximately 2000-fold less than the peak serum IgG1 levels induced by GaM delta injection. Both IgE and IgG1 secretion in GaM delta injected mice were T dependent (blocked by anti-L3T4 antibody). These observations are consistent with the hypothesis that BSF-1 may play a role in the in vivo stimulation of IgE secretion and provide an easy to apply model for the investigation of in vivo regulation of IgE responses.     

Anti-IgD enhancement of primary antibody responses in rats

The role of membrane IgD in immune responses was examined by treating adult rats with anti-IgD. Anti-IgD when administered to rats in conjunction with optimal or suboptimal doses of either SRBC, a T-dependent antigen, or DNP-Ficoll, a T-independent antigen, enhanced the antibody responses. The greatest enhancement was obtained when anti-IgD was administered before the antigen. The effects of anti-IgD on antibody responses to SRBC were: (i) significant antibody responses to suboptimal antigen concentrations; (ii) greater antibody responses to optimal antigen concentrations; (iii) accelerated antibody responses; (iv) an early shift from IgM to IgG antibodies; (v) prolonged antibody responses. Similar effects on the immune response to DNP-Ficoll were observed with the exception that all antibodies were 2ME sensitive (IgM). These results suggest that an anamnestic type of immune response can be induced in anti-IgD-treated rats when given a primary antigen exposure. Injection of anti-IgD without SRBC or DNP-Ficoll induces B-cell proliferation without detectable antibody production to these antigens, indicating at least two signals are required for the enhanced antibody responses.     

Nippostrongylus brasiliensis: radioresistant IgE antibody-forming cells in infected rats

In Nippostrongylus brasiliensis-infected rats, anti-N. brasiliensis IgE antibody production was observed at 20 weeks postinfection, long after the worms, as a source of antigen, had been expelled. The persistent IgE production was not abrogated after whole body irradiation (800 R) administered at 12 or 20 weeks, suggesting the participation of radioresistant IgE-forming cells. Help of T cells and recruitment of B memory cells in the irradiated rats seems to be ruled out by the findings that the irradiation completely inhibited the initiation of anti-N. brasiliensis IgE production in rats shortly after the infection with N. brasiliensis or after primary and secondary immunization with N. brasiliensis-antigen. Moreover, clearance of anti-N. brasiliensis IgE antibody from circulation did not seem to be crucially affected by the irradiation. The radioresistant cells forming anti-N. brasiliensis IgE were most productive in mesenteric lymph nodes as compared to other lymph nodes. The recognition of antigens fractionated by chromatography on Sephadex G-200 was the same for IgE-forming cells from rats 12 weeks after infection as for those from 3 weeks after infection. Based on these results, one of the mechanisms of persistent elevation of IgE antibody in the host infected with helminth parasites might be explained by the participation of radioresistant IgE-forming cells.     

The IgE and IgG subclass responses of mice to four helminth parasites

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.     

Epitope-specific regulation. IV. In vitro studies with suppressor T cells induced by carrier/hapten-carrier immunization

Sequential immunization with a carrier molecule and a new epitope (hapten) conjugated to the carrier (carrier/hapten-carrier immunization) induces specific suppression for IgG antibody production to the new epitope (hapten) on the carrier. Once induced, this "epitope-specific" suppression persists and specifically suppresses subsequent in vivo IgG antibody responses to the hapten presented on the same or on an unrelated carrier molecule. In vitro studies presented here characterize the surface markers and specificity of suppressor T cells generated in carrier/hapten-carrier-immunized animals. Thus we show (1) that spleen cells from these donors suppress in vitro IgG anti-hapten antibody production by cocultured hapten-primed spleen cells; (2) that some but not all of the suppressor cells carry surface Lyt-2; (3) that at least some of the suppressor cells have receptors for the inducing hapten (DNP); and (4) that, unlike the suppression obtained in vivo, the in vitro suppression extends to IgG responses to unrelated carrier protein epitopes presented in association with the inducing hapten.     

7S class-restricted hapten-specific paralysis by injection of thymus-independent hapten-carrier conjugate.

The shift from IgM anti-hapten antibody production to IgG anti-hapten antibody production, subsequent to challenge with a T-dependent conjugate, is inhibited in mice by preimmunization with the T-independent DNP-LE conjugate. The results suggest that DNP on levan triggers off 19A T-independent anti-hapten precursors and paralyzes 7S T-dependent anti-hapten precursors.     

IL-4 is required to generate and sustain in vivo IgE responses

Antibodies of the IgE isotype play a predominant role in immediate hypersensitivity reactions. IL-4, a T cell-derived lymphokine that stimulates increased Ia expression by resting B cells and increased IgG1 secretion by LPS-activated B cells in vitro, has also been shown to regulate in vitro and in vivo polyclonal IgE responses. We report that large quantities of a purified anti-IL-4 mAb inhibit primary in vivo polyclonal IgE responses by 99% in mice infected with Nippostrongylus brasiliensis or injected with anti-IgD antibodies, and totally inhibit secondary Ag-specific IgE responses to TNP-keyhole limpet hemocyanin without effect on either IgG1 or IgG2a responses to these stimuli. The lack of effect of anti-IL-4 antibody on IgG1 secretion cannot be explained simply by inadequate neutralization of IL-4, inasmuch as the doses of anti-IL-4 antibody used blocked an N. brasiliensis-induced increase in B cell Ia expression by more than 85%, whereas in vitro studies indicate that enhancement of B cell Ia expression requires less IL-4 than induction of IgG1 secretion. In addition to demonstrating that IL-4 plays a necessary role in the generation of an in vivo IgE response, we show that IL-4 has an important role in sustaining established IgE responses, because anti-IL-4 antibody, when given at the peak of an N. brasiliensis- or TNP-keyhole limpet hemocyanin-induced IgE response, accelerates the declines in total serum IgE and in IgE anti-TNP antibody levels, respectively. These observations suggest that the effects of IL-4 on in vivo immune responses may be more specific than might have been predicted from in vitro observations, and that regulation of IL-4 production or action may be useful for the prevention or therapy of immediate hypersensitivity disorders.     

Enhancement of antigen-induced interleukin 4 and IgE production by specific IgG1 in murine lymphocytes.

Conalbumin (CA)-specific type 2 helper T cell (Th2) clone, D10G4.1 (D10) produces IL4 when stimulated with varying doses of TNP-CA in the presence of mitomycin C-treated C3H spleen cells or purified B cells as antigen-presenting cells (APC). The production of IL4 was assessed by bioassay and by expression of IL4 mRNA. IL4 production reached maximum at 100 micrograms/ml of TNP-CA, whereas 1 microgram/ml of the antigen induced less than 10% of the maximum level of IL4. This lower level of IL4 production was augmented to the maximum level when monoclonal anti-TNP IgG1 was added to the culture at 0.5-1 microgram/ml. Anti-TNP IgE, but not anti-TNP IgM, was also effective, though IgE was 1/10 as effective as IgG1. IgG1 with an irrelevant specificity and F(ab')2 of anti-TNP IgG1 did not show augmenting effects. Moreover, the enhancement by anti-TNP IgG1 was completely abolished by monoclonal antibody against murine Fc gamma RII, 2.4G2. These results suggest that a low dose of the antigen complexed with IgG1 is focused on APC by means of Fc gamma RII, processed, and presented efficiently to the Th2 clone. On the other hand, the co-culture of D10 with normal C3H B cells in the presence of 1-100 micrograms/ml TNP-CA resulted in polyclonal IgE production. Anti-TNP IgG1 markedly augmented the lower level of IgE production induced by a suboptimal dose of the antigen (1 microgram/ml). This augmentation was shown to be dependent on endogenous IL4 because the enhancement was abolished by monoclonal anti-IL4 (11B11).     

Anti-hapten IgG antibodies bound to cell surface hapten: anti-IgG antibody prevents dissociation as measured with fluid phase hapten

We investigated the dissociation by fluid phase hapten of IgG antibodies bound to cell surface hapten in the presence and absence of anti-IgG antibodies. Dissociation was quantitated with fluid phase hapten, preventing reassociation of the anti-hapten antibodies. More than 90% of the anti-hapten IgG alone was prevented from reassociation by low concentrations of fluid phase hapten (nanogram to microgram range). In contrast, no dissociation of some IgG-anti-IgG complexes could be measured even at 24 hr incubation in the presence of very large excess of fluid phase hapten (100 mg/ml). We excluded aggregate formation between anti-hapten antibodies due to cross-linking by anti-antibodies as a cause for decreased dissociability by 1) performing the experiments in large excess of anti-antibody, 2) showing that the phenomenon was independent of anti-hapten antibody density, 3) showing that decreased dissociation also occurred at 4 degrees C, and 4) showing that aggregation by protein A did induce decreased dissociability, albeit three orders of magnitude lower than the anti-antibody. It was concluded that anti-antibody directly affected the "avidity" of cell hapten bound anti-hapten IgG in an unknown manner.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.