Failure of rIL-12 administration to inhibit established IgE responses in vivo is associated with enhanced IL-4 synthesis by non-B/non-T cells
Julia D. Rempel1,
MingDong Wang and
Kent T. HayGlass
Department of Immunology, University of Manitoba, 626730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada
Correspondence to:
K. HayGlass
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Abstract
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Administration of rIL-12 offers a widely successful tactic for preferential induction of type 1 immune responses in vivo. Its use to modulate ongoing cytokine or effector responses has proven to be substantially more difficult. Immediate hypersensitivity is the most common human immunologic disease. Here, rIL-12 was administered to C57Bl/6 and outbred CD1 mice with ongoing ovalbumin (OVA)-specific IgE responses in an attempt to redirect established type 2 cytokine and antibody production. Despite use of a broad range of treatment protocols for >4 months following initial immunization, recall IgE responses were consistently unaffected. rIL-12-treated mice exhibited strong in vivo and in vitro IFN-
responses, increased ~40-fold relative to controls, but also markedly enhanced (15- to 20-fold) OVA-specific IL-4 production. CD4 T cell function was successfully transformed from a type 2- to a type 1-dominated pattern following long-term IL-12 administration in vivo, as measured by strongly reduced IL-4 and IL-10 responses in antigen-stimulated primary culture, and 5-fold reductions in the frequencies of IL-4- and IL-10-producing OVA-specific CD4 T cells. However, chronically rIL-12-treated mice exhibited increased numbers of non-B/non-T cells that when re-stimulated with specific allergen, produce IL-4 at levels 20-fold higher than did CD4 T cells while IL-13 responses are unaffected. Collectively, the data indicate that even effectively shifting CD4 T cell activation from a type 2- to a type 1-dominated response does not in itself lead to altered effector (IgE) responses upon antigen re-exposure.
Keywords: IL-4, IL-12, immediate hypersensitivity, non-B/non-T
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Introduction
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Allergic diseases represent the most common, and one of the most rapidly increasing, human immunologic disorders (1). Excessive expression of type 2 cytokine synthesis is of pivotal importance in their pathogenesis and maintenance (2,3). Consequently, much effort has been devoted to strategies which might allow re-direction of Th2-dominated responses to Th1-like patterns.
IL-12 is produced by macrophages, dendritic cells and other antigen-presenting cells, but not normally B cells, in response to various activators, including bacterial stimuli, intracellular parasites and exogenous antigen (46). Endogenous IL-12 production plays a key role in shaping initiation of type 1 and type 2 responses (79). Moreover, exogenous IL-12 co-administered with antigen can steer immune activation such that normally dominant Th2-like responses develop as Th1-like responses. Thus, when rIL-12 is administered at the time of initial immunization, it results in responses characterized by increased IFN-
and IgG2a synthesis, and decreased primary IL-4 and IgE. A broad variety of disease models have been examined with generally similar results (4,1016).
The observation that IL-12 is an effective inhibitor of T cell-dependent induction of IgE secretion by IL-4-stimulated human peripheral blood mononuclear cells (17), taken with reports that in vitro addition of rIL-12 to memory CD4+ cells from allergic patients decreased IL-4 and IL-10 synthesis (18), and that successful allergen immunotherapy is associated with enhanced endogenous IL-12 expression (19), have stimulated interest in the potential therapeutic utility of exogenous rIL-12 administration in immediate hypersensitivity diseases. However, the few studies examining the capacity of rIL-12 to redirect ongoing responses in vivo have generally proven to be unsuccessful.
Here we examined the ability of rIL-12, used in inbred and outbred murine strains under a range of experimental protocols, to redirect pre-established type 2 responses. We report that independent of the number of ovalbumin (OVA) immunizations or courses of exogenous cytokine administered, IL-12 strongly enhanced OVA-specific type 1 responses, yet consistently failed to inhibit specific IgE production. The failure to abrogate established IgE responses was not attributable to an inability to redirect the OVA-specific T cell response. Limiting dilution analysis demonstrated a shift from Th2-dominated cytokine responses to a Th1-like pattern with marked reductions in the frequency of OVA-specific IL-4- and IL-10-producing CD4 T cells and a change in the ratio of type 2:type 1 cytokine responses from 7.2 to 0.6. However, these changes in the antigen-specific T cell response were overwhelmed by exogenous IL-12-driven expansion of IL-4-producing non-B/non-T cells, resulting in a net 20-fold enhancement of antigen-dependent IL-4 synthesis in IL-12-treated mice. The data suggest that while long-term administration of rIL-12 to individuals with established IgE responses may ultimately re-direct de novo CD4 T cell populations from type 2- to type 1-dominated patterns, this is overwhelmed by increased antigen-specific IL-4 synthesis contributed by non-B/non-T cell populations, with the net result that serum IgE levels in vivo are not inhibited.
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Methods
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Mice
C57Bl/6 and CD1 mice were bred at the University of Manitoba breeding facility (Winnipeg, Canada) or purchased from Charles River Canada (St Constant, Quebec, Canada) and were used in accordance with guidelines issued by the Canadian Council on Animal Care.
Immunization and treatment protocol
Primary immunization consisted of 0.2 or 2 µg OVA (5 times recrystallized; ICN, Montreal, Canada) adsorbed onto 2 mg Al(OH)3 adjuvant (alum) given i.p. on day 0. Mice were subsequently immunized and treated with rIL-12 (a gift from Dr Maurice Gately, Hoffmann-La Roche, Nutley, NJ) using various protocols as described in Results. In all experiments the final OVA boost, given a minimum of 28 days following the preceding exposure to antigen to allow the previous response to subside, was accompanied by a course of IL-12 administration running from the day prior to immunization and continuing daily over the next 5 days. IL-12 was administered at 200 ng/mouse i.p. in saline containing 0.4% normal mouse serum. Preliminary experiments carried out using 5001000 ng/injection of rIL-12 resulted in intense splenomegaly (data not shown and 2022) leading us to evaluate this cytokine at the maximum concentration that did not lead to toxicity. Mice were bled 7 days after each immunization for total and OVA-specific serum antibody determinations.
Cell culture
In most experiments, mice were sacrificed 5 days after final immunization, the time of peak secondary cytokine responses (data not shown). Duplicate spleen cell suspensions from two to three mice per group per time point were cultured and analyzed independently. For bulk primary culture, cells were cultured at 7.5x106/ml (2 ml/well) alone or with OVA as reported (21). Culture of CD4-enriched cells (9296% CD4+ by flow cytometry) was carried out using 106 responder cells/200 µl well, with 3x105 irradiated naive spleen cells as a source of APC and OVA. Culture of `non-B/non-T cells' was carried out under the same conditions in 200 µl wells using 500,000 flow cytometry negatively selected cells/well, also in the presence and absence of OVA. Unless stated otherwise, in most experiments culture supernatants were harvested for analysis of IL-4, IFN-
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IL-10 and, in some experiments, IL-13 production at 24, 48 and 96 h time points was previously found to correspond to optimum cytokine levels (21,24). Analysis of cytokine production by non-B/non-T cells was carried out at 3, 24, 48 and 72 h; 24 h results are shown.
Limiting dilution analysis
Five days after final immunization, control and rIL-12-treated mice were sacrificed. Spleen cell suspensions, pooled within each group, were panned on goat anti-mouse Ig-coated Petri plates to deplete adherent and sIg+ cells. The non-adherent populations were harvested and passed through a CD4 T cell negative selection column (Biotex, Edmonton, Canada), yielding 9296% CD4+ cells with <0.7% contamination with B cells or CD8+ T cells as assessed by subsequent flow cytometry. This population was serially diluted (312.5 to 40,000 cells/well; 36 replicate wells per dilution) and cultured in round-bottom 96-well culture plates (Corning Science Products, Corning, NY) with 3x105 irradiated spleen cells (1500 rad), 20 U/ml rIL-2 (Chiron, Seattle, WA) and OVA. Each plate also included 12 wells of irradiated naive spleen cells in the absence of responder cells as negative controls. To evaluate potential spontaneous cytokine production, rarely observed control cultures were also established for high responder cell concentrations (10,000 to 40,000 cells/well) under identical conditions except for the absence of OVA. Cultures were incubated 14 days, at which time proliferation was visually assessed under low-level magnification (x10). All cells were then washed and re-stimulated with fresh irradiated APC (3x105) and OVA (21). Supernatants were harvested at 48 h, and assayed for IFN-
, IL-10 and IL-4 production. In each assay, wells were considered positive for cytokine production if the absorbance obtained in a given well was >3 SD above the mean value obtained in wells containing APC, rIL-2 and antigen in the absence of CD4 cells (negative control, carried out on each plate).
Cytokine and antibody determinations
Cytokines.
IL-4 levels were determined in an MTS assay using CT.4S cells as previously described (24), and, where indicated, were confirmed by ELISA using capture and development reagents purchased from PharMingen. IFN-
, IL-10 (PharMingen, San Diego, CA) and IL-13 (R & D Systems, Minneapolis, MN) responses were determined in ELISA using cytokine specific mAb as previously reported (23). Assay SEM were typically <5%. Detection limits were typically 0.2 U/ml for IL-4, 0.15 U/ml for IFN-
, 0.3 U/ml for IL-10 and 5 pg/ml for IL-13.
IgE.
OVA-specific IgE production was determined by 48 h passive cutaneous anaphylaxis (PCA) in female Sprague-Dawley rats as described (25). Geometric means of duplicate or triplicate independent analyses are presented. Total serum IgE levels were determined by ELISA. Briefly, ELISA plates (25805; Corning) were coated overnight at 4C with 2 µg/ml of rat anti-mouse IgE (Southern Biotechnology Associates, Birmingham, AL) in bicarbonate buffer (0.05 M, pH 9.6). After blocking 45 min at 37C with a 1% BSA/0.05% Tween 20 solution and washing, four to eight dilutions were incubated for 2 h at 37°C. Detection was with a biotinylated epsilon specific monoclonal rat anti-mouse IgE heavy chain (Serotec, Oxford, UK). Highly purified anti-DNP mouse IgE, prepared from hybridoma 2682 (a gift of Dr A. Sehon, University of Manitoba), was used as a standard. This assay has a sensitivity of 0.4 ng/ml.
IgG1 and IgG2a.
OVA-specific murine IgG1 and IgG2a were determined in an alkaline phosphatase-based ELISA as previously described (25), using four to eight dilutions for each sample, calibrated against a polyclonal murine anti-OVA standard. Total murine IgG1 and IgG2a were determined in subclass specific ELISAs developed using sheep anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) at 2 µg/ml to capture and biotinylated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates) followed by streptavidinalkaline phosphatase. Internal standards of IgG1 and IgG2a from B cell hybridomas, calibrated against mouse IgG2a,
(UPC 10; Sigma, St Louis, MO) and a purified anti-OVA IgG1 mAb generated by Dr G. Lang (University of Manitoba, Winnipeg, Canada). The sensitivity of these assays was typically 0.5 ng/ml for IgG1 and 0.1 ng/ml for IgG2a.
Flow cytometry.
To determine the impact of IL-12 administration on the proportion of different cell populations within the spleen, 0.5x106/well were incubated with anti-mouse CD32/CD16 (PharMingen, San Diego, CA) for 15 min followed by FITC-conjugated rat IgG2a anti-mouse CD4, CD8 and/or CD19 (PharMingen) at 1.5, 1.5 and 3.0 µg/106 cells respectively. Control samples were incubated with FITC-conjugated rat anti-mouse IgG2a at 1.5 µg/0.5x106 cells (PharMingen) for 45 min at 4°C. Non-B/non-T populations were isolated by negative selection using a protocol based on that previously reported (26,27). Briefly, spleen cells were incubated on ice with FITC-conjugated anti-mouse CD4, CD8 and CD19 as described above, with the unstained cells negatively selected by flow cytometry. The resulting negatively selected population contained <5% contamination. Flow cytometric analysis and cell sorting were performed using a Beckman Coulter (Miami, FL) Epics 753 cell sorter with an argon ion laser emitting 500 mW at 488 nm. Instrument alignment and performance were assessed using DNA-Check fluorospheres (Beckman Coulter) in terms of coefficient of variation (CV) and target channel numbers for linear forward angle light scatter and fluorescence signals yielding CVs in the range of 0.550.70%. All fluorescence histograms were of 256 channel resolution, based on log amplified (three-decade) signals and at least 5000 gated events. Control histograms derived from cells treated with FITC-conjugated rat IgG2a were employed to establish the sort windows for `non-stained' cells and included 9095% of all events. Subsequently, spleen cells labeled with anti-mouse CD4, CD8 and CD19 were sorted using these criteria to isolate unstained non-B/non-T cell populations. Re-analysis of the sorted populations indicated >95% purity.
Statistical analysis
For each ELISA described, data were obtained from a titration of at least four dilutions against the standard curve. OVA-specific IgE data were log2 transformed and analyzed as geometric means. Because outbred mice were used, statistical significance for antibody and serum IFN-
levels was evaluated for B6 and outbred mice by the MannWhitney test. Analysis of cytokine responses, carried out for C57Bl/6 only, were performed with the unpaired two-tailed Student's t-test. Estimates of the precursor frequency of CD4 T cells reactive to OVA were obtained by both the maximum likelihood and minimum
2 methods based on the Poisson distribution relationship between the number of responding cells and the logarithm of the fraction of negative cultures. Software used was generously provided by Dr C. Orosz (Ohio State University, Columbus, OH). The results obtained were similar using either method. Maximum likelihood results are presented.
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Results
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IL-12 enhances antigen-specific IgG2a synthesis but consistently fails to abrogate established IgE responses
To evaluate the capacity of exogenous IL-12 to alter ongoing IgE responses, a number of regimens were examined. Mice were challenged and boosted with 2.0 µg OVA in 10 experiments or, in four experiments, 0.2 µg, the lowest concentration of OVA consistently able to elicit specific IgE responses. C57Bl/6 and, as representatives of genetically diverse populations, outbred CD1 mice were exposed to treatment protocols ranging from a single course of five 200 ng rIL-12 injections administered immediately prior to secondary OVA immunization (which ranged from day 21 to 88) to five courses of rIL-12 administered over alternate weeks beginning 4.5 months after a single OVA booster. With each of the approaches taken, exogenous IL-12 failed to significantly inhibit expression of recall OVA-specific IgE responses.
In the examples shown, B6 (Table 1
) mice were immunized, rested 4 months to ensure that secondary IgE responses had subsided and then treated with five courses of IL-12 (day 168229), each consisting of five injections each of 200 ng rIL-12 and were then re-challenged with OVA.
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Table 1. IL-12 administration to C57B1/6 mice with established type 2 responses markedly enhances OVA-specific IgG2a production but fails to inhibit IgEa
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For outbred CD1 mice, Table 2
shows the data obtained when IL-12 was administered beginning shortly after (30 day) a single priming immunization with OVA. Under each of the protocols tested, OVA-specific secondary IgE responses were indistinguishable in IL-12-treated and control groups. Indeed, total serum IgE was 2- to 3-fold increased, particularly in long-term rIL-12-treated mice (Table 1
). IL-12 administration also enhanced median specific and total IgG1 levels by ~2-fold in most instances. In contrast, OVA-specific IgG2a responses in IL-12-treated B6 mice were strongly increased, ranging from 50- to 150-fold in B6 and 5- to 20-fold in CD1 mice. A similar picture, with striking increases in IgG2a production paralleled by unchanged to 2- to 3-fold enhanced IgE/IgG1 responses, was found in 14 independent experiments examining a variety of other treatment protocols (data not shown). Thus, administration of extended courses of exogenous IL-12 to mice with established type 2 responses strikingly enhanced the antigen-specific component of the type 1-dependent humoral immune response but was unable to inhibit expression of recall type 2 antibody responses.
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Table 2. IL-12 administration to outbred mice with established responses enhances OVA-specific IgG2a synthesis but fails to inhibit IgE recall responsesa
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In vivo administration of IL-12 elevates serum IFN-
The capacity of IL-12 to strongly promote type 1 responses independent of its failure to abrogate established type 2 antibody responses was further demonstrated by examination of serum IFN-
levels (Fig. 1
). rIL-12-treated B6 and outbred mice both exhibited enhanced serum IFN-
upon antigen re-exposure. This response peaked at days 45 with IFN-
levels in all groups returning to pre-immunization levels by day 7 (<2 U/ml, data not shown). These data indicate that the inability of IL-12 to abrogate secondary IgE responses was not due to a failure to induce intense systemic IFN-
production in vivo.
Differential effects of in vivo IL-12 administration on heterogeneous spleen populations and CD4 T cells
To evaluate the impact of rIL-12 administration on cytokine production during secondary responses, primary cultures were established. As for serum IFN-
, in vivo administration of IL-12 enhanced OVA-stimulated IFN-
responses in culture by an average of 13-fold and reduced IL-10 synthesis by a similar margin (Fig. 2
). Surprisingly, IL-4 production in antigen-driven bulk culture was 15- to 30-fold elevated. Control cultures carried out without OVA exhibited minimal cytokine levels, regardless of whether the mice received rIL-12 or not (data not shown).
To better characterize these changes, OVA-stimulated spleen cell populations or, in parallel, highly enriched CD4 spleen cells were cultured in the presence of irradiated naive spleen cells as APC (Table 3
). Unseparated spleen cell populations from rIL-12-treated mice exhibited strongly increased OVA-driven IFN-
and IL-4 responses (mean ~35- and 20-fold increases over the four experiments performed) and substantially decreased IL-10 synthesis (~15-fold relative to immunized controls not treated with IL-12). In contrast, cytokine production by CD4 T cell-enriched populations isolated from the same donors exhibited no increases in cytokine synthesis. As previously, cytokine production in cultures without OVA stimulation were at or below the limits of detection.
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Table 3. rIL-12 administration to mice with established type 2 responses yields distinct effects on spleen cells and enriched CD4 T cell populationsa
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This unexpected result was independently confirmed through limiting dilution analysis. Enriched CD4 T cells, taken from the same individual donors used for the bulk cultures shown in Table 3
, were used to assess the impact of rIL-12 treatment on the frequency of OVA-specific CD4 T cells in mice with ongoing type 2 responses. As demonstrated in Table 4
, repeated courses of rIL-12 consistently failed to alter the frequency of IFN-
-producing OVA-specific CD4 cells. The decrease in IL-10 synthesis seen in whole spleen culture was directly reflected in reductions in the activity and the frequency of the IL-10-producing, OVA-specific CD4 population studied in bulk culture (Table 3
) and limiting dilution analysis (Table 4
). Of greatest importance was the median 6-fold decrease in the frequency of IL-4-producing OVA-specific CD4 cells that resulted from chronic IL-12 treatment. Thus, following extended rIL-12 treatment, the Th1:Th2 balance seen in the OVA-specific CD4 T cell repertoire was reversed from type 2 dominance (median IL-4:IFN-
ratio of 7.2:1 among OVA-specific CD4 T cells in the absence of IL-12 administration) to one characterized by type 1 dominance (median IL-4:IFN-
ratio of 0.6:1 in rIL-12-treated mice).
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Table 4. IL-12 administration to mice with established IgE responses reduces the frequency of CD4 T cells producing Th2-associated cytokinesa
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Notwithstanding this change in OVA-specific CD4 T cell frequencies, the net OVA-driven response, specifically the balance of IL-4:IFN-
production seen with heterogeneous spleen cells rather than highly enriched CD4 T cells, was essentially unaffected despite extended courses of IL-12 administration (whole spleen data from Table 3
). Taken together, the data argue that in vivo rIL-12 administration results in a marked shift in the OVA-specific CD4 T cell response that is effectively overwhelmed by the impact of IL-12 on other cells capable of producing large amounts of IL-4 in response to antigen-specific re-stimulation.
rIL-12 administration enhances the frequency and function of non-B/non-T cells
Non-B/non-T cells from individuals with ongoing immediate hypersensitivity responses have been shown in animal and human systems to be potent producers of IL-4 (2730). We, therefore, directly examined the frequency of such cells in spleens of mice immunized and treated as described above. As demonstrated by flow cytometry analysis of spleen cells from mice with ongoing type 2 responses that received rIL-12 treatment, the number and proportion of non-B/non-T cells in the spleen, defined as CD4CD8CD19 cells, was 100300% enhanced relative to OVA-immunized/non-IL-12-treated controls (Fig. 3
). The proportion of CD4 cells was reduced upon IL-12 treatment but the actual number remained essentially unchanged. There was no significant change in the frequency of CD8 or CD19 cells.
To evaluate the capacity of such cells to produce type 2 cytokines IL-4 or IL-13 in response to OVA-mediated stimulation, non-B/non-T-enriched populations were negatively selected by flow cytometry using antibodies to CD4, CD8 and CD19, and cultured alone or with OVA at a range of concentrations. As seen in Fig. 4
, mean IL-4 production by non-B/non-T cells from immunized mice treated with IL-12 was substantially greater than that seen in OVA-immunized controls and required substantially less antigen (10 versus 3001000 µg/ml for CD4 T cell activation, data not shown) to elicit cytokine synthesis. IL-13 production was substantial but did not differ between the two groups under the conditions tested. Naive mice or those treated with rIL-12 in the absence of OVA immunization failed to produce substantive quantities of cytokine. All of the groups produced IL-4 and IL-13 at or below the assay detection limits in the absence of OVA re-stimulation. Collectively, the data indicate that the pronounced increases in the capacity of these mice to mount IL-4 responses, and 2- to 3-fold increases in IgE synthesis, are associated with a capacity for exogenous IL-12 to both stimulate expansion of the numbers of non-B/non-T cells in vivo and to strikingly enhance IL-4 production by those cells in response to antigen-mediated re-stimulation.

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Fig. 4. CD4CD8CD19 cells from IL-12-treated mice with ongoing type 2-dominated responses exhibit markedly increased production of IL-4, but not IL-13 in response to antigenic stimulation. Negatively selected cells from spleens of naive, naive/rIL-12-treated, OVA-immunized or OVA-immunized/IL-12-treated mice were cultured with medium alone (all cytokine levels below limits of detection, data not shown) or with OVA at the indicated antigen concentrations for 24 h. Mean (±SEM) IL-4 and IL-13 levels in culture supernatants were determined by ELISA as described at Methods. Data shown represent one of three similar experiments.
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Discussion
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We report that in vivo administration of exogenous IL-12, given under a variety of conditions, consistently fails to abrogate established IgE responses. This inability to modulate recall type 2 antibody production is not attributable to a failure to alter the frequency of antigen-specific CD4 T cells, which were shifted from a median Th2:Th1 ratio of 7:1 to one of 0.6:1 following chronic IL-12 administration, but to selective expansion and activation of IL-4-producing non-B/non-T cells, that were responsible for a net 20-fold increase in OVA-stimulated IL-4 synthesis. We speculate that IL-12-enhanced IL-4 synthesis plays an important role in maintaining IgE levels in these mice, despite the activation of strong splenic and serum IFN-
responses and marked enhancement of type 1 (IgG2a) antibody responses in vivo.
The finding that in vivo administration of rIL-12 leads to marked enhancement of IL-4 synthesis has an in vitro parallel. Freshly isolated, neonatal human CD4 T cell lines derived in the presence of rIL-4 + rIL-12 were found to produce IL-4 and IFN-
at levels that were substantially increased relative to those derived in the absence of exogenous rIL-12 (31). The current report, demonstrating greatly increased numbers and type 2 cytokine synthesis by CD4CD8CD19 cells in response to in vivo rIL-12 administration, makes it clear that enthusiasm for in vivo utilization of nominally type 1-promoting cytokine or chemokines for therapeutic purposes (reviews 3235) needs to be viewed with caution.
A potential trivial explanation for our failure to inhibit ongoing IgE responses in vivo is that the amount of rIL-12 administered was too small or the time course too short. While this remains a possibility, we believe it unlikely for several reasons. Mice were treated with IL-12 using protocols that ranged from a single course of injections over 5 days, begun immediately after the primary IgE response began to subside (day 21), to repeated series of six courses of five injections each over a period ending 230 days later. Decreasing the amount of OVA to the smallest amount of antigen capable of consistently eliciting primary IgE responses (0.2 µg) also failed to improve the capacity of IL-12 to redirect effector responses. Indeed, the only consistently observed change in type 2 antibody production was a 2- to 3-fold enhancement in total IgE production. It remains possible that administration of substantially higher amounts of exogenous IL-12, or IL-12 given in association with other recombinant cytokine or chemokine, would be effective in preventing specific recall IgE responses (36), but this would need to be balanced against the splenomegaly and potential toxicity associated with such levels (2123). Approaches such as transient mucosal IL-12 gene delivery using vaccinia (37) or adenovirus (38) vectors may be capable of re-directing ongoing antigen-specific cytokine and IgE responses from type 2- to type 1-dominated patterns, but the impact of such approaches on the net cytokine response and resulting clinical status remain to be determined.
We initially hypothesized that rIL-12 administration to mice with established type 2 responses would increase the precursor frequency of OVA-specific CD4+ cells producing IFN-
and, perhaps, reduce the frequency of IL-4 producers. However, notwithstanding the major increase in IFN-
synthesis observed in vivo (serum IFN-
) and in vitro (bulk culture of antigen stimulated intact spleen cell populations), a finding that is likely associated with NK cell activation (21,3741), we found that even prolonged courses of rIL-12 administration had no detectable impact on the frequency of CD4 T cells secreting IFN-
. This finding, in this model of ongoing responses, parallels that made in IL-12 pre-treatment models (21) where exogenous IL-12 administration elicited intense but transient Th1-like effects that were primarily NK cell dependent and were not associated with increases in the frequency of IFN-
producing, antigen-specific CD4 T cells.
While endogenous IL-12 plays a pivotal role in shaping the development of immune responses in naive individuals, the effects of exogenous IL-12 administration remain controversial. The capacity of IL-12 to influence T cell activation in already differentiated systems is greatly impaired (4,8,9). Similarly, the later that rIL-12 is added in vitro, the less impact it has on cytokine synthesis (4,6). Thus, as primed T cells become conditioned, its ability to modify cytokine profiles diminishes. These findings are consistent with the observed stability of most differentiated Th2 clones where at early stages they respond to IL-12 with transient IFN-
(and undiminished IL-4) synthesis (42,43), while well-established Th2 clones lose the capacity to respond to IL-12 due to loss of IL-12 receptor ß2 (44,45). Thus, our finding that extended in vivo administration of IL-12 to mice with well-established IL-4/IgE responses leads to markedly lower frequencies of OVA-specific IL-4-producing CD4 T cell populations is of particular interest. We believe the key distinction is that studies of IL-12's ability to alter established Th2 clones (or fresh Th2-like T cells) in vitro generally focus on fully differentiated T cell responses. Our efforts to alter Th2-associated cytokine and antibody responses in vivo target both (i) lymphocyte populations that have already established stable commitment to a Th2-like response weeks or months earlier (and may be largely resistant to change) and (ii) de novo commitment of naive T cells taking place over the term of the experiment. The results of the CD4 T cell limiting dilution analyses indicate that long-term IL-12 treatment of individuals with intense ongoing type 2 responses sharply reduces commitment of naive T cell populations to type 2 patterns despite the presence of a well-established antigen-specific IL-4 response in vivo.
At the same time, these data caution that even qualitative alterations in the Th1/Th2 balance of the CD4 T cell repertoire (i.e. reversal of the IL-4:IFN-
ratio from frequencies of 7:1 to 0.6:1) can be overwhelmed by other IL-12-induced changes, such as enhanced non-B/non-T cell-mediated IL-4 production, that could severely limit the clinical utility of this strategy. Indeed, populations enriched in CD4CD8CD19 cells, taken from mice with ongoing type 2 responses (with IgE responses consistently equivalent in all groups, independent of whether IL-12 treatment was given) exhibited OVA-specific IL-4 production substantially greater than that did CD4 T cell populations obtained from donors in the same experiment (Table 3
).
There is limited information on the impact of in vivo IL-12 treatment on established type 2 responses to protein antigen. Germann et al. (46), using bee venom derived phospholipase A2 and keyhole limpet hemocyanin (KLH), evaluated a comprehensive range of in vivo treatment protocols utilizing rIL-12. When given with initial immunization, IL-12 strongly suppressed primary IgE responses. However, established responses were transiently inhibited, unchanged or somewhat increased depending on the particular conditions tested. Cytokine production was not evaluated. Bliss et al., studying established IgE responses to TNP-KLH in BALB/c mice, were also unable to abrogate recall IgE synthesis. They reported enhanced IL-4 synthesis following primary culture of unseparated spleen cells from IL-12-treated mice and speculated that its source was CD4 T cells (47). In both these studies, the reasons for the failure of IL-12 to redirect the balance of the immune response were not investigated.
Detailed characterization of the non-B/non-T cell responsible for enhanced IL-4 synthesis upon antigen-specific re-stimulation remains to be accomplished. One reasonable candidate is the basophil (2730, 48), but the nature and existence of murine basophils remains controversial. A second potential candidate is the NK T cell (4952). Flow cytometric analysis shows minimal increases in the number of NK1.1+, CD3+CD4CD8 cells in the spleens of rIL-12-treated mice (Solomon et al., unpublished data). Recent data indicate that IL-18, a potent amplifier of type 1 immunity, can also promote markedly enhanced IL-4 and/or IL-13 synthesis by basophil, NK cells and T cells (48, 52). A specialized population of 
T cells is also capable of robust IL-4 production (53), although in each of these instances the means by which antigenic specificity for nominal antigen would be conferred remains speculative. The precise nature of the CD4CD8CD19 population(s) described in this report that exhibits strikingly increased frequency and IL-4-secreting activity upon IL-12 treatment, as well as the means by which it is activated to produce IL-4 in an antigen-dependent fashion, is currently under active investigation.
In summary, these data demonstrate that rIL-12 given after development of IgE responses fails to redirect established cytokine and antibody responses from type 2-dominated patterns. While prolonged rIL-12 administration efficiently shifts the balance of CD4 T cell function to a type 1-dominated pattern by reducing Th2-like CD4 T cell frequencies and cytokine synthesis, it also results in markedly enhanced numbers of CD4CD8CD19 cell populations that produce markedly enhanced antigen-dependent IL-4 responses. The net result of these changes is induction of Th1-like effects (enhanced IFN-
, IgG2a synthesis) concomitant with strikingly enhanced IL-4 and total IgE synthesis.
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Acknowledgments
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We thank Bill Stefura for dedicated technical assistance, and Dr Ed Rector for expert advice and assistance with flow cytometry. This work was supported by the Medical Research Council of Canada. J. R. held a Manitoba Health Research Council Studentship; K. H., an MRC Scientist award.
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Abbreviations
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APC antigen-presenting cell |
KLH keyhole limpet hemocyanin |
OVA ovalbumin |
PCA passive cutaneous anaphylaxis |
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Notes
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1 Present address: Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA 
Transmitting editor: K. Takatsu
Received 9 September 1999,
accepted 15 March 2000.
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References
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