Differentiation Initial Source of IL-4 During Th2 T Cells Provide an +

Conventional, Naive CD4+ T Cells Provide an Initial Source of IL-4 During Th2 Differentiation

This information is current as Nancy Noben-Trauth, Jane Hu-Li and William E. Paul of September 27, 2021. J Immunol 2000; 165:3620-3625; ; doi: 10.4049/jimmunol.165.7.3620 http://www.jimmunol.org/content/165/7/3620 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Conventional, Naive CD4؉ T Cells Provide an Initial Source of IL-4 During Th2 Differentiation

Nancy Noben-Trauth,1 Jane Hu-Li, and William E. Paul

IL-4 is known to promote the differentiation of CD4؉ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor ␣-chain -IL-4R␣؊/؊) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a require) ment for prior IL-4-mediated stimulation. To address whether naive, conventional CD4؉ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4R␣؊/؊mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4R␣؊/؊ DO11.10/scid mice secreted ϳ50 pg of IL-4 per106 cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4؉ T cells into Th2 cells, particularly if IFN-␥ and ؉ ␣؊/؊ ϳ IL-12 were neutralized during the priming cultures. CD4 cells from the IL-4R mice gave rise to a minor proportion ( 2%) Downloaded from of IL-4-producing cells upon stimulation in the presence of anti-IFN-␥ and anti-IL-12. These data show that conventional, naive CD4؉ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-␥ and IL-12, this IL-4 can induce Th2 polarization. The Journal of Immunology, 2000, 165: 3620–3625.

L-4 is a major inducer and mediator of allergic and parasitic pacity of these cells to become IL-4 producers in the absence of immune responses. One major mechanism through which it added IL-4 and concluded that, depending on the concentration of http://www.jimmunol.org/ mediates this function is in promoting the differentiation of Ag and the degree of costimulation, Th2 priming could be I ϩ naive CD4 T cells into Th2 cells (1–5). The identiﬁcation of the achieved (18, 19, 22). In some experiments, it was shown that such initial cellular source of the IL-4 that mediates Th2 cell induction priming was blocked by anti-IL-4. is key to designing therapies to intervene in the development of These studies, although arguing that naive cells can be the Th2 responses. Based on prior studies, cells that have been iden- source of IL-4, do not conclusively establish this point. Most im- tiﬁed to secrete IL-4 include cells of the mast cell/basophil lineage portantly, the cells were drawn from mice that can both make and (6–9), eosinophils (10, 11), NK1.1ϩ, CD4ϩ T cells (NK T cells)2 respond to IL-4 so that such cells could have received an IL-4 (12–14), ␥/␦ T cells (15), and conventional CD4ϩ T cells (16–22). signal in vivo and differentiated into Th2 cells and then returned to

Conventional T cells, either those that have been previously the resting state. Such cells would then be able to make IL-4 upon by guest on September 27, 2021 primed or naive cells, are attractive candidates as a source of the restimulation. Indeed, because the TCR transgenic mice used were “early” IL-4 because they are in the “right place at the right time.” not on scid or RAG backgrounds, their expression of alternative TCR Whether such cells could be prime movers has been uncertain ␣-chains makes environmental immunization possible. Furthermore, because their acquisition of the ability to produce IL-4 has gener- it is now clear that CD62L is not a reliable marker of the naive state ally been regarded as an IL-4-dependent event. However, it has because memory cells can revert to being CD62L bright (23). been suggested that naive T cells may make small amounts of IL-4 We have readdressed this important issue using mice in which (or rare naive T cells may make large amounts) simply in response the IL-4R␣-chain has been disrupted, crossed to TCR transgenic to TCR/CD28-mediated stimuli. To establish this, one needs to use mice on a scid background, and have further used IL-4ϫIL-4R␣ highly purified populations of naive, conventional CD4ϩ T cells double-knockout (KO) cells as APC. The possibility of ␥/␦ or NK and be assured that these cells have not previously received an T cells as a source of IL-4 is eliminated in this system because IL-4 receptor-mediated signal. neither cell type is present in these mice. Neither basophils/mast Previous groups have made efforts to establish the capacity of cells nor eosinophils are likely to be involved because few, if any, naive T cells to produce the early IL-4 needed for priming. In of these cells are found in lymph nodes (LNs), which were our general, they have used cells with high buoyant density that were source of T cells. Finally, because we use APCs from IL-4ϫIL- sorted for expression of high levels of CD62L or CD45RB and/or 4R␣ donors, the APC can neither produce nor consume IL-4. This low levels of CD44. In some cases, this has been done in mice that makes a source of IL-4 other than the naive T cells most unlikely were TCR transgenic. These studies generally measured the ca- and also allows us to quantitate IL-4 production because depletion of IL-4 from culture medium by receptor-mediated endocytosis should be largely eliminated. This system allows an unambiguous Laboratory of Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 measure of the early production of IL-4 by conventional, naive T Received for publication February 23, 2000. Accepted for publication July 12, 2000. cells and a determination of whether this IL-4 is sufficient to prime naive IL-4R␣ϩ cells to become Th2 cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 Address correspondence and reprint requests to Dr. Nancy Noben-Trauth, Labora- Mice tory of Immunology, National Institutes of Health, Twinbrook II, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address: [email protected] BALB/c IL-4Ϫ/Ϫ and IL-4R␣Ϫ/Ϫ mice were generated as described (24, 2 Abbreviations used in this paper: NK T cells, NK1.1ϩ, CD4ϩ T cells; WT, wild- 25) and bred under specific pathogen-free conditions in the National In- type; LN, lymph node; cRPMI, complete RPMI; KO, knockout; L, ligand. stitute of Allergy and Infectious Diseases Animal Care Unit. DO11.10/scid

TCR mice (26) were crossed to the IL-4R␣Ϫ/Ϫ mice and screened by Results FACS for the TCR clonotype KJ1-26 (Caltag, South San Francisco, CA) Conventional T cells from IL-4R␣Ϫ/Ϫ mice produce an early and CD45R/B220 (PharMingen, San Diego, CA) expression on PBLs and for the presence of the mutated IL-4R␣ locus by PCR. 5CC7ϫRAG2 TCR burst of IL-4 Ϫ/Ϫ (27) mice and IL-4 mice on a B10.A background were bred at Taconic To address whether naive conventional CD4ϩ T cells are capable Farms (Germantown, NY). The BALB/c nude mouse strain (nu/nu) was purchased from the Division of Cancer Treatment, National Cancer Insti- of producing an initial burst of IL-4 upon TCR, we crossed tute (Frederick, MD). DO11.10 TCR transgenic mice on a homozygous scid background to the BALB/c IL-4R␣Ϫ/Ϫ mutants. Such mice would lack NK T Culture medium cells, and the scid mutation would guarantee exclusive expression of the appropriate transgenic TCR. To further exclude IL-4 pro- Complete RPMI (cRPMI) consisted of RPMI 1640 medium (Biofluids, duction from the splenic APC population and consumption of the Rockville, MD) supplemented with 10% FBS (Life Technologies, Rock- L IL-4 produced in the cultures, we used irradiated T-depleted spleen ville, MD), 1 mM sodium pyruvate, 2 mM -glutamine, 0.05 mM 2-ME, Ϫ Ϫ 100 U/ml penicillin, and 100 ␮g/ml streptomycin. cells from (IL-4ϫIL-4R␣) / double-deficient mice as APC. LN cells from DO11.10/scid IL-4R␣ϩ/Ϫ and IL-4RaϪ/Ϫ mice Abs and cytokines were cultured with 3 ␮M OVA peptide, and supernatants were 6 ␥ ␤ obtained at 48 h (Fig. 1). Such supernatants contained 50 pg/10 CD4-CyChrome, IL-4-PE, IFN- -FITC, V 8-FITC, anti-CD3, and anti- ␣ϩ/Ϫ IL-3 were all purchased from PharMingen. Human IL-2 was a gift from cells/ml of IL-4. We failed to detect any IL-4 when IL-4R Perkin-Elmer/Cetus (Norwalk, CT). Anti-IL-4 (11B11), anti-IL-12 cells were cultured with peptide alone. However, the addition of (C17.8), anti-IFN-␥ (XMG 1.2), and anti-mouse Fc␥ receptor (2.4G2) anti-IL-4R␣ mAb resulted in the appearance of a comparable Downloaded from mAbs were all purified from ascites by Harlan Biosciences (Madison, WI). amount of IL-4 in supernatants from such cells, arguing that the ␣ Anti-mouse IL-4R (M1) was purchased from Genzyme (Cambridge, “early” IL-4 was rapidly consumed by the CD4ϩ T cells. Thus, MA). Goat anti-mouse IgD plasma was a generous gift from Fred Finkel- ␣ϩ/Ϫ ␣Ϫ/Ϫ man (University of Cincinnati, OH). Mouse IL-4 was obtained from a both IL-4R and IL-4R DO11.10 cells respond to OVA, recombinant baculovirus prepared by Cynthia Watson (Laboratory of Im- resulting in the production of 50 pg of IL-4 per million cells within munology, National Institute of Allergy and Infectious Diseases). Recom- 48 h. binant mouse IL-3 was purchased from Genzyme. For T cell depletions, ϩ spleen cells were incubated with 20% supernatants of anti-Thy-1.2 mAb IL-4 made by conventional CD4 T cells can result in Th2 http://www.jimmunol.org/ HO13.4 and rabbit complement (Cedarlane Laboratories, Hornby, Ontario, polarization Canada). To assess whether the small amount of IL-4 produced within 48 h ELISAs after stimulation was capable of generating a Th2 response, LN cells from DO11/10/scid/IL-4R␣ϩ/Ϫ and IL-4R␣Ϫ/Ϫ mice were IL-4 (Endogen, Woburn, MA) and IL-2 (PharMingen) ELISAs were performed according to manufacturer directions. For the serum IgE ELISA, stimulated for a 5-day priming period with OVA peptide in the 96-well plates were coated with 2 ␮g/ml each of two monoclonal anti-IgE presence of irradiated T-depleted spleen cells from IL-4ϫIL- Abs (02131D from PharMingen and AMI2501 from BioSource, Camarillo, 4R␣Ϫ/Ϫ donors. Amounts of IL-4 in the supernatants and the fre- CA). After blocking and overnight incubation with serum samples, plates quency of IL-4-producing cells were measured after restimulation by guest on September 27, 2021 were developed with HRP-conjugated goat anti-IgE Abs (Southern Bio- with peptide and APCs (Fig. 2 and Table I). technology Associates, Birmingham, AL) followed by peroxidase substrate (Bio-Rad, Hercules, CA). The mAb IgE standard (D-8406) was purchased When no other additions were made to the initial priming cul- from Sigma BioSciences. ture, the frequency of IL-4-producing cells was at the borderline of detectability (Fig. 2, “peptide alone”) and the amounts of super- In Vitro priming natant IL-4 were low (Table I). The addition of IL-4 to the IL- ϩ/Ϫ ϩ Ϫ 4R␣ cells dramatically increased the frequency of IL-4-pro- Mesenteric LN cells (1 ϫ 106/ml) from DO11.10/scid IL-4R␣ / and IL- 4R␣Ϫ/Ϫ mice were cultured in 24-well plates in cRPMI and IL-2 (10 U/ml) ducing cells and the amount of supernatant IL-4. As expected, no Ϫ/Ϫ and stimulated with 3 ␮M OVA peptide and T-depleted irradiated spleen change was seen in the IL-4R␣ cells cultured with IL-4. cells (5 ϫ 106/ml) from BALB/c IL-4ϫIL-4R␣ double-deficient mice as APC. The peptides for OVA (323–339) and pigeon cytochrome c (88–104) were purchased through National Institute of Allergy and Infectious Dis- eases, Laboratory of Molecular Structure-Peptide Synthesis Laboratory. Some cultures also contained anti-IFN-␥ (10 ␮g/ml) and anti-IL-12 (10 ␮g/ml), IL-4 (1000 U/ml), or anti-IL-4 (20 ␮g/ml). On the second or third day of culture, the cells were diluted 1:5 in cRPMI with IL-2 and expanded into 6-well plates. Five to six days after the initial stimulation, cells were harvested, washed twice, and replated at 1 ϫ 106/ml in cRPMI with IL-2 and challenged for an additional 5-day priming under the same conditions. LN cells from 5CC7ϫRAG2 TCR transgenics were primed similarly, but with 3 ␮M PCC peptide and T-depleted irradiated spleen cells (5 ϫ 106/ ml) from B10.A IL-4Ϫ/Ϫ mice as APC.

Intracellular cytokine staining In vitro primed cells were harvested, washed, and cultured at 1 ϫ 106/ml FIGURE 1. Ag-specific “early” IL-4 secretion in DO11.10/scid mice. in cRPMI in 48-well plates with 3 ␮M OVA peptide, 3 ␮M monensin LN cells from DO11.10/scid IL-4R␣ϩ/Ϫ and IL-4R␣Ϫ/Ϫ mice were stim- 6 (PharMingen), and 5 ϫ 10 /ml T-depleted irradiated spleen cells from ulated at 1 ϫ 106/ml in 24-well plates with 3 ␮M OVA peptide for 48 h in ϫ ␣ Ϫ/Ϫ BALB/c (IL-4 IL-4R ) mice as APC. After 6 h, cells were washed, cRPMI containing 10 U/ml human IL-2. Anti-IL-4R␣ (10 ␮g/ml) was fixed in 4% paraformaldehyde, and permeabilized with 0.1% saponin in added to some cultures. T cells were depleted from splenic cultures by PBS containing 1% FCS. Cells were first blocked with 2.4G2 (10 ␮g/ml) then stained simultaneously with CyChrome-anti-CD4, FITC-anti-IFN-␥, complement-mediated lysis as described in Materials and Methods. APCs ϫ ␣ Ϫ/Ϫ ϫ 6 and PE-anti-IL-4 (all obtained from PharMingen) and analyzed by FACS. from BALB/c (IL-4 IL-4R ) mice were included at 5 10 /ml to LN For the 5CC7ϫRAG2 culture, cells were stimulated with 3 ␮M PCC pep- cultures. The amounts of IL-4 and IL-2 in the 48-h supernatants were tide, 3 ␮M monensin, and T-depleted irradiated spleen cells (5 ϫ 106/ml) measured by ELISA. The results are representative of three independent from B10.A IL-4Ϫ/Ϫ mice. experiments. 3622 IL-4 SECRETION BY NAIVE T CELLS

FIGURE 2. In vitro priming of DO11.10/scid/IL- 4R␣ cells. A, LN cells from DO11.10/scid IL-4R␣ϩ/Ϫ and IL-4R␣Ϫ/Ϫ mice were stimulated for 5 days under the indicated conditions at 1 ϫ 106/ml in 24-well plates containing 3 ␮M OVA peptide and T-depleted spleens from BALB/c (IL-4ϫIL-4R␣)Ϫ/Ϫ mice as APC. B, Cells were subjected to an additional round of peptide priming under the same conditions. For the analysis, cells were washed, and 1 ϫ 106 cells were restimulated with 3 ␮M OVA peptide and 5 ϫ 106 (IL-4ϫIL- 4R␣)Ϫ/Ϫ APCfor6hinthepresence of 3 ␮M monensin. Cells were stained with IL-4-PE, IFN-␥-FITC, and

CD4-CyChrome. The results shown are gated on the Downloaded from CD4ϩ populations and are representative of two experiments. http://www.jimmunol.org/

If IL-4R␣ϩ/Ϫ cells were cultured without added IL-4 but in the tion of the responding cells (i.e., the IL-4R␣ϩ/Ϫ cells) when IFN-␥ presence of anti-IFN-␥ and anti-IL-12, 4% of the cells produced and IL-12 are neutralized. IL-4, and supernatant IL-4 content was 4823 pg/106 cells. When When these cells were recultured for five additional days under anti-IL-4 Ab was added to such cultures, the number of IL-4- the same conditions, they proved to be excellent IL-4-producers by guest on September 27, 2021 secreting cells fell to 1.7%, and the amount of supernatant IL-4 when challenged; 29.3% produced IL-4 (Fig. 2B), and supernatant was reduced to 1121 pg/106 cells. This argues that the endogenous IL-4 content was 157,000 pg/106 cells (Table I). Only 4.2% of the production of IL-4 by IL-4R␣ϩ/Ϫ cells was sufficient to cause the cells primed in the presence of anti-IL-4, anti-IL-12, and anti- induction of a modest number (ϳ3%) of IL-4-producing cells but IFN-␥ produced IL-4 after the second round of priming. This only if IFN-␥ and IL-12 were neutralized. strongly implies that the enhanced IL-4 production observed after The data also show that there is a small number IL-4-producers the second round of stimulation is in response to endogenously even in the absence of stimulation through the IL-4 receptor. The produced IL-4, and not simply due to repetitive stimulation. latter conclusion is supported by results with the IL-4R␣Ϫ/Ϫ cells In contrast to the wild-type (WT) cells, the twice-primed IL- primed in the presence of anti-IFN-␥ and anti-IL-12, with or with- 4R␣Ϫ/Ϫ cells had a frequency of IL-4-secreting cells ranging from out IL-4 or anti-IL-4. In these cultures, supernatant IL-4 levels 2.2–3.0%, and the amount of supernatant IL-4 ranged from 1600– ranged from 386–646 pg/106 cells, slightly less than that obtained 2140 pg/106 cells in the cultures containing anti-IFN-␥ and anti- in the IL-4R␣ϩ/Ϫ cells cultured with anti-IFN-␥, anti-IL-12, and IL-12. The latter provides a more rigorous reflection of the IL-4- anti-IL-4 (1121 pg/106 cells) (Table I). independent component of the polarization process than the result These results lead us to conclude that the modest amount of IL-4 obtained with heterozygous cells. produced by DO11/10/scid/IL-4R␣ϩ/Ϫ and Ϫ/Ϫ CD4ϩ T cells in Because these cells are derived from BALB/c mice, the possi- response to Ag stimulation is sufficient to polarize a small propor- bility remained that the IL-4 response we observed was peculiar to

Table I. IL-4 levels (picograms per milliliter) in cultures from in vitro primed DO11.10/scid/IL-4R␣ WT and KO LN, and 5CC7 LN cellsa

LN Donor Priming Peptide Alone IL-4 IL-4, anti-IFN-␥, anti-IL-12 Anti-IL-4, anti-IFN-␥, anti-IL-12 Anti-IFN-␥, anti-IL-12

IL-4Rϩ/Ϫ One round 271 94,493 88,639 1,121 4,823 IL-4RϪ/Ϫ 121 181 646 386 540 5CC7 64 23,368 19,403 456 379

IL-4Rϩ/Ϫ Two rounds 630 329,000 95,348 3,305 91,281 IL-4RϪ/Ϫ 146 293 1,600 2,140 1,703 5CC7 Not done 125,000 131,000 1,119 17,094

a LN cells (1 ϫ 106/ml) from DO11.10/scid IL-4R␣ϩ/Ϫ and IL-4R␣Ϫ/Ϫ mice were stimulated in 24-well plates with 3 ␮M OVA peptide for 5–6 days with T-depleted spleens from BALB/c (IL-4 ϫ IL-4R␣)Ϫ/Ϫ as APC under the conditions indicated and described in Fig. 2. For the analysis, cells were washed, and 1 ϫ 106 cells were restimulated with 3 ␮M OVA or pigeon cytochrome c peptide and 5 ϫ 106 (IL-4 ϫ IL-4R␣)Ϫ/Ϫ APC. IL-4 was measured by ELISA in supernatants taken at 24 h. For the 5CC7 cultures, LN cells were stimulated with 3 ␮M pigeon cytochrome c peptide and T-depleted spleens from B10.A IL-4Ϫ/Ϫ mice as APC. The Journal of Immunology 3623

Table II. Cell recoveries (ϫ106) in cultures from in vitro primed DO11.10/scid/IL-4R␣ WT and KO LN, and 5CC7 LN cellsa

LN Donor Priming Peptide Alone IL-4 IL-4, anti-IFN-␥, anti-IL-12 Anti-IL-4, anti-IFN-␥, anti-IL-12 Anti-IFN-␥, anti-IL-12

IL-4Rϩ/Ϫ One round 9.5 10.8 19.0 16.3 25.6 IL-4RϪ/Ϫ 14.6 12.7 24.6 27.6 24.4 5CC7 13.3 8.6 16.8 15.8 18.6

IL-4Rϩ/Ϫ Two rounds 7.5 9.5 12.5 9.2 13.0 IL-4RϪ/Ϫ 8.3 6.9 12.9 10.3 9.8 5CC7 not done 5.0 13.1 7.4 9.5

a LN cells (1 ϫ 106/ml) from DO11.10/scid IL-4R␣ϩ/Ϫ and IL-4R␣Ϫ/Ϫ mice and 5CC7 ϫ RAG2 mice were stimulated in 24-well plates as described in Fig. 2 and Table I. Cells were recovered after one and two rounds of in vitro priming, and viable cells were counted by trypan blue exclusion. this mouse strain, which has been reported to “default” to Th2 cells after each round of priming. There was no appreciable difference upon priming in the absence of added IL-4 (28). To address this, in either IL-4R␣ϩ/Ϫ or Ϫ/Ϫ groups cultured with or without IL-4 we performed a similar experiment with cells from 5CC7ϫRAG2 (Table II). This makes it quite unlikely that the enhanced differ- TCR transgenic mice, which are on a B10.A background. These entiation of IL-4R␣ϩ/Ϫ cells into IL-4 producers can be accounted cells were primed with 3 ␮M PCC peptide. As shown in Fig. 2, the for by a proliferation mechanism. Downloaded from 5CC7 cells showed trends in IL-4 production comparable to the ϩ BALB/c IL-4R␣ϩ/Ϫ mice. More importantly, a small percentage Anti-IgD injection results in IL-4 production by CD4 T cells ␣Ϫ/Ϫ of IL-4 producers were detected after the first round of priming from IL-4R mice with pigeon cytochrome c peptide in the presence of anti-IFN-␥ Our results indicate that naive CD4ϩ T cells secrete an early burst and anti-IL-12. This was strikingly augmented after the second of IL-4 that is capable of driving Th2 responses in vitro. We next priming. This further confirms that vigorous Th2 responses de- asked whether such IL-4R-independent IL-4 production is func- ϩ velop similarly in CD4 T cells from BALB/c and B10.A back- tionally active during an in vivo response. A potent stimulus of http://www.jimmunol.org/ grounds, particularly when IFN-␥ and IL-12 are neutralized. IL-4 production and of IgE responses is treatment of mice with Thus, in the absence of exogenous IL-4, a limited amount of goat Ab to mouse IgD (30). Although the stimulus is specific for IL-4 is produced by naive cell populations; such production is B-cells, Ag-specific CD4ϩ T cells (30) are required to induce the sufficient so that a substantial degree of polarization can be response. It has also been shown that NK T cells do not contribute achieved through repetitive culture. Endogenously produced to IL-4 production because CD1Ϫ/Ϫ mice that are deficient for NK IFN-␥ and IL-12 will block this effect, presumably by limiting the T cells retain IL-4-dependent IgE responses after anti-IgD treat- responsiveness of the T cells to the very modest amounts of IL-4 ment (31). produced early in the first culture. To determine whether the capacity of T cells to produce IL-4 in by guest on September 27, 2021 IL-4 has been shown to enhance survival of T and B cells (as response to anti-IgD required prior priming through the IL-4 re- reviewed in Ref. 29). To exclude the possibility that the increase in ceptor, WT and IL-4R␣Ϫ/Ϫ mice were treated with anti-IgD. As ϩ Ϫ IL-4-production in the IL-4R␣ / cell population compares to IL- shown in Fig. 3A, the frequency of cells with cytosolic IL-4 in Ϫ Ϫ 4R␣ / cells reflects preferential survival of the IL-4R␣-express- response to anti-IgD was comparable between the heterozygous ing cells, we measured the total number of live cells recovered controls and IL-4R␣Ϫ/Ϫ mice. Nearly all IL-4 producers were in

FIGURE 3. Conventional CD4ϩ T cells produce IL-4 after anti-IgD treatment. IL-4R␣ϩ/Ϫ and IL-4R␣Ϫ/Ϫ mice were injected s.c. with 200 ␮l FIGURE 4. IL-4 produced by anti-IgD treated IL-4R␣Ϫ/Ϫ mice can of goat anti-mouse IgD plasma. Spleens were removed at 5 days, stimu- functionally drive IgE class switching. BALB/c and IL-4R␣Ϫ/Ϫ mice were lated with 10 ng/ml of PMA, 500 ng/ml of ionomycin, and 3 ␮M monensin injected s.c. with 200 ␮l of goat anti-mouse IgD plasma. Spleen cells (40 ϫ (PharMingen) for 6 h, and analyzed for intracellular IL-4 production by 106) from BALB/c nu/nu mice were injected i.v. into IL-4R␣Ϫ/Ϫ mice 2 cytoplasmic staining and flow cytometry. Top, IL-4 production by total days after anti-IgD injection according to the method described (45). Mice CD4ϩ T cells. Bottom, The frequency of V␤8ϩ IL-4-producing cells after were bled 8 days after anti-IgD treatment, and total serum IgE levels were gating on CD4ϩ cells. Untreated controls shown were from IL-4R␣ϩ/Ϫ measured by ELISA. The results are from three mice per group Ϯ SD and mice. represent two individual experiments. 3624 IL-4 SECRETION BY NAIVE T CELLS the CD4ϩ T cell population. In addition, using quantitative, com- cient to prime for a delayed, but eventually quite striking, Th2 petitive RT-PCR, the number of IL-4 transcripts in total spleen response, if IL-12 and IFN-␥ are neutralized. Ϫ Ϫ cells from anti-IgD treated IL-4R␣ / mice was actually 2-fold These experiments also show that a small percentage of trans- greater than that of WT mice (data not shown). genic CD4ϩ T cells from IL-4R␣Ϫ/Ϫ mice can acquire IL-4-pro- ϩ The IL-4-producing CD4 T cells do not appear to be from the ducing capacity when primed in the absence of IFN-␥ and IL-12. subset of NK T cells; the proportion of IL-4-producing cells from Whether this represents IL-4-independent in vitro priming or the Ϫ/Ϫ ϩ the IL-4R␣ mice that are V␤8 is 21% (1.5 of 7.2% total IL-4 response of cells that were actually primed in vivo is not certain. producers), which is only slightly greater than the frequency of The latter is rendered unlikely by the fact that the cells used were ␤ ϩ V 8 cells that did not produce IL-4 (13.5%) (Fig. 3B). NK T derived from mice on a scid background, which should be much ␤ ϩ cells have been shown to have a frequency of V 8 cells of less likely to be primed by environmental Ags. ϳ ϩ 55%. Thus, it seems likely that conventional CD4 T cells can Our results demonstrate that there is an IL-4-independent in be stimulated by anti-IgD to produce IL-4 even if these cells are vivo pathway of IL-4 secretion by “conventional” T cells. Injection incapable of responding to IL-4. of anti-IgD into IL-4R␣ mutant mice resulted in the same fre- Because the capacity of conventional CD4ϩ T cells from IL- quency of IL-4-producing CD4 T cells as in WT mice (Fig. 3). 4R␣Ϫ/Ϫ mice to produce IL-4 in response to anti-IgD injection, we Although Kaplan et al. have reported that that IL-4-secreting cells next asked whether this IL-4 was functionally capable of stimu- Ϫ Ϫ in STAT6 / mice express the NK Ag DX5, it is most unlikely lating B cell Ig class switching to IgE, which is generally an IL- that the cells making IL-4 in response to anti-IgD are NK T cells. 4-dependent event. We transferred WT spleen cells from BALB/c Ϫ/Ϫ IL-4 production in response to anti-IgD occurs normally in NK T ␣ Downloaded from nu/nu mice to IL-4R recipients and asked whether anti-IgD Ϫ/Ϫ Ϫ/Ϫ could induce IgE expression in the reconstituted animals. As cell-deficient mice (CD1 and B2 m ) (31, 34), and our anal- ␤ ϩ ␣Ϫ/Ϫ shown in Fig. 4, anti-IgD-treated IL-4R␣Ϫ/Ϫ mice had very low ysis of the frequency of V 8 CD4 T cells in the IL-4R mice levels of IgE, but when reconstituted with nu/nu spleen cells, they makes it unlikely that the IL-4 producers are NK T cells (Fig. 3). developed IgE levels of 60.4 ␮g/ml, equivalent to the response of Further evidence for the IL-4-independent appearance of IL-4- ␣Ϫ/Ϫ Ϫ/Ϫ intact BALB/c mice. It is interesting to note that even without the producing Th2 cells is from a study in IL-4R and in Stat6 mice infected with Schistosoma mansoni. CD4ϩ T cells from these addition of WT spleen cells, IgE was modestly increased in the http://www.jimmunol.org/ ϩ IL-4R␣Ϫ/Ϫ mice after anti-IgD treatment (undetectable to 308 ng/ mice produce IL-4, although at a lower frequency than do CD4 ml). IL-4-independent IgE production has been observed in other T cells from infected WT mice (35). Careful analysis of the CD4 systems; in IL-4 and IL-4R␣ KOs, IgE production of ϳ0.1–1% of cells from the mutant mice and of schistosome egg Ag-specific T normal is often observed (32). Thus, anti-IgD injection induced cell clones derived from them showed that the cells had the ca- IL-4R␣-independent IL-4 production by a cell population that can pacity to produce IL-5 as well as IL-4, strengthening the conten- mediate Ig class switching, further supporting the notion that these tion that they were authentic Th2 cells. are “conventional” CD4ϩ T cells. Another in vivo system in which IL-4-independent induction of Th2 responses occurs is in BCL6Ϫ/Ϫ mice. These mice develop a Discussion severe Th2-type myocarditis and pulmonary vasculitis, marked by by guest on September 27, 2021 It is clear that in vivo and in vitro, IL-4 plays a major role in the eosinophil infiltration and by the production of IL-4, IL-5, IL-10, induction of Th2 responses (1–5, 33). The cellular source of this and IL-13 (36). BCL6 has been shown to be capable of opposing “inducing” IL-4 has been a matter of great interest that has been Stat6-induced transcription of some genes (36); nonetheless, a sim- investigated by several groups. In this report, we contribute to ilar syndrome occurs in (BCL6ϫSTAT6)Ϫ/Ϫ mice, indicating that these studies by quantitating the early burst of IL-4 occurring after in the absence of BCL6, a STAT6-independent pathway of prim- ϩ ␣Ϫ/Ϫ initial Ag stimulation of CD4 IL-4R T cells. Cells from ing for IL-4 production is revealed (37). ␣ IL-4R mutant mice provide a unique opportunity to measure such Ouyang and colleagues have recently reported that a proportion early IL-4 production because IL-4 is not consumed by these cells, of DO11.10 TCR transgenic STAT6Ϫ/Ϫ cells develop into IL-4- nor can IL-4-dependent, IL-4-producing cells appear in the culture. producers when primed in vitro (38). They demonstrated that in ␣ Furthermore, the absence of IL-4R assures that there is no pos- such cells GATA3 mRNA levels were elevated, and that the sibility that these cells would be previously exposed to IL-4 in vivo DNase I hypersensitivity had been induced in the second intron of and therefore “primed”, either by IL-4 produced by endogenously the IL-4 gene, consistent with the concept that the pathway of Th2 activated CD4ϩ cells or by non-T cell sources of IL-4 such as mast priming, although IL-4-independent, was still GATA3 dependent. cells. This result and the results from the (BCL6ϫSTAT6)Ϫ/Ϫ mice are We examined Ag-specific IL-4 production by LN cells obtained consistent with the existence of a single common pathway for Th2 from D011.10/scid mice that were either mutant or wild type at the differentiation involving GATA3. This pathway is most efficiently IL-4R␣ locus. Under these conditions, ϳ50 pg/ml were produced ␣ by 106 cells. This is an amount that is less than that required to stimulated through IL-4R /STAT6 activation, but it may also be optimally polarize naive CD4ϩ T cells to become Th2 cells (gen- induced through some other mechanism(s), possibly through TCR erally 500-1000 pg/ml) but may still be sufficient to cause a degree and accessory molecule engagement with appropriate APCs. Such of polarization. IL-4-independent priming has been reported when human DC2 Therefore, we asked whether T cells from WT DO11.10/scid dendritic cells, but not DC1 cells, have been used to stimulate ϩ mice could use their endogenously secreted IL-4 to differentiate naive (CD45RO ) CD4 T cells to become IL-4 producers (39). into Th2 cells. Our data demonstrate that LN cells from DO.11.10/ Furthermore, it has been reported that APCs that fail to express scid mice stimulated with 3 ␮M OVA peptide prime for IL-4 pro- ICAM-1 efficiently induce Th2 responses (40, 41). ϩ duction when IFN-␥ and IL-12 were neutralized. Under these con- Thus, these results indicate that some conventional CD4 T ditions, ϳ4% of the T cells became IL-4 producers. When cells can produce IL-4 in response to Ag-mediated T cell activa- stimulated through a second round, this rose to 30%, presumably tion, and that such IL-4 can then drive the bulk of the responding due to the substantial amounts of IL-4 produced in the first round. CD4 T cells to differentiate into polarized Th2 cells. The factors Thus the early IL-4 secreted by naive T cells appears to be suffi- that govern the initial production of IL-4 are a complex interplay The Journal of Immunology 3625 of peptide dose, costimulatory molecules, the strength and repeti- 21. Demeure, C. E., L. P. Yang, D. G. Byun, H. Ishihara, N. Vezzio, and tion of the stimulus, and the presence of down-regulatory cyto- G. Delespesse. 1995. 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