International Immunology

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International Immunology, Vol. 11, No. 2, 243-247, February 1999
© 1999 Japanese Society for Immunology

Marking IL-4-producing cells by knock-in of the IL-4 gene

I-Cheng Ho^1,2, Mark H. Kaplan¹, Laurie Jackson-Grusby³, Laurie H. Glimcher^1,2 and Michael J. Grusby^1,2

¹ Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA
² Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
³ The Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA

Correspondence to: M. J. Grusby, Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA

	Abstract

Top Abstract Introduction Methods Results and discussion References

 
IL-4 is a cytokine which can be expressed by a number of cell types including T_h2 cells, mast cells and a population of CD4⁺ NK1.1⁺ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-4-secreting T_h2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells.

Keywords: cytokines, T_h1/T_h2, transgenic/knockout

	Introduction

Top Abstract Introduction Methods Results and discussion References

 
IL-4 is a 20 kDa glycoprotein originally identified by its ability to support the growth and differentiation of B lymphocytes co-stimulated with submitogenic doses of anti-Ig (1). IL-4 is now known to elicit a wide variety of responses by multiple cell types. For example, IL-4 has been shown to promote the differentiation of B lymphocytes and their switch to the production of IgE and IgG1 isotypes (2,3), and to be important for the development of cytotoxic T lymphocytes (CTL) (4,5). In addition, IL-4 is critical for the differentiation of T_h2 cells (6,7).

IL-4 was initially described as a T cell-derived cytokine (8) and its expression is one of the criteria used to distinguish T_h2 cells from IFN--secreting T_h1 cells (9). Like many cytokines, however, IL-4 is now recognized to be produced by multiple cell types. For example, IL-4 has been shown to be secreted by mast cells (10), CTL (11,12) and T cells (13). In addition, a population of CD4⁺ NK1.1⁺ NK T cells has been shown to promptly produce large amounts of IL-4 following TCR ligation in vivo (14).

Each of these IL-4-producing cell types can be identified by the expression of specific cell surface markers. For example, NK T cells are often identified by their expression of CD4 and NK1.1, while T cells are defined by their expression of the TCR. Recently, several cell surface markers have been identified which show selective expression on T_h2, but not T_h1 cells, including the eotaxin receptor CCR3 (15) and a molecule designated ST2L (16). Unfortunately, there is at present no known cell surface marker common to all IL-4-producing cell types. Moreover, it is not clear if the expression of molecules such as CCR3 and ST2L exactly correlates, on a cell to cell basis, with IL-4 expression in cells in vivo. While IL-4-producing cells can be identified by intracellular cytokine staining and flow cytometry, these procedures do not allow for the recovery of viable cells. Given these constraints in the ability to detect and manipulate those cells which are actually producing IL-4, we have used gene targeting in embryonic stem cells to modify the IL-4 locus and generate mice that express a membrane-bound form of IL-4 (mIL-4). Using an IL-4-specific mAb, we were able to detect IL-4-secreting T_h2 cells, mast cells and NK T cells from mIL-4 mice by flow cytometry. Furthermore, the analysis of immune responses in mIL-4 mice has allowed us to identify distinct subpopulations of IL-4-producing NK T cells.

	Methods

Top Abstract Introduction Methods Results and discussion References

 
Generation of membrane-bound IL-4 mice
The targeting construct used to generate a knock-in of the IL-4 locus contained exons 3 and 4 of the IL-4 gene, and was bounded by the EcoRI site located 1 kb upstream of exon 3 and the XbaI site located 3 kb downstream of exon 4 (17). Exon 4 of the murine IL-4 gene was modified by recombinant PCR to contain a transmembrane anchor. Briefly, the fourth exon of the MHC class II A_ß^b gene (18), encoding the connecting peptide, the transmembrane region and the first six amino acids of the cytoplasmic tail, was inserted between the last codon and the translational stop site of the IL-4 gene. A cassette containing the neomycin resistance gene was placed 150 nucleotides downstream of the polyadenylation signal of the IL-4 gene. The targeting construct was transfected into day 3 embryonic stem cells as described (19). Homologous recombinants were identified by Southern analysis of EcoRI-digested DNA using the 3' probe shown in Fig. 1; this probe detects a 7 kb fragment of the wild-type allele and a 4.5 kb fragment of the mIL-4 allele. One homologous recombinant was used to generate chimeras that passed the mIL-4 allele through the germline. Mice were genotyped using the primers shown in Fig. 1 and having the sequence: IL-4 upstream, 5'-GAG ACC CAA ATC TGT CTC AC-3'; IL-4 downstream, 5'-GTT AAA GCA TGG TGG CTC AG-3'. Unless otherwise noted, all experiments were done on 3- to 4-month-old homozygous mIL-4 mice which had been backcrossed six generations onto the BALB/c genetic background.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Generation of mIL-4 Mice. (Top) The endogenous IL-4 locus (top), the targeting construct (middle) and the predicted structure of the mIL-4 allele (bottom). (Bottom) PCR analysis of offspring resulting from heterozygous intercross of mIL-4 mice.

 
In vitro differentiation of T_h2 cells
Spleen cells from wild-type and mIL-4 mice were stimulated in vitro with 1 µg/ml plate-bound anti-CD3 (2C11) and 20 µg/ml anti-IL-12 (5C3). Twenty-four hours later, 50 U/ml IL-2 and 500 U/ml IL-4 were added. Seven days post-stimulation, cells were harvested, washed and re-stimulated with plate-bound anti-CD3 for 24 h. Cells were then stained with FITC-conjugated anti-IL-4 (11B11) (PharMingen, San Diego, CA) and analyzed by flow cytometry.

Preparation and stimulation of peritoneal mast cells
Mouse peritoneal mast cells were prepared as described (20). Briefly, the peritoneal cavity was lavaged with 10 ml modified Tyrode's buffer (2.7 mM KCl, 12 mM NaHCO₂, 0.4 mM NaH₂PO₄, 0.14 M NaCl, 0.1% glucose, 0.1% gelatin). The recovered peritoneal cells were washed once, resuspended in modified Tyrode's buffer (1 ml/pooled cells from 10 mice), layered onto a solution of 22.5% metrizamide and centrifuged for 12 min at 1500 r.p.m. The cell pellet was harvested, washed twice and used as a source of enriched mast cells. Enriched mast cells were then stimulated with 1 µM ionomycin for 6–8 h, stained with FITC-conjugated anti-IL-4 and analyzed by flow cytometry.

In vivo immunization
Anti-CD3 (100 µg per animal in 100 µl of PBS) or PBS was administered i.v. by injection into the tail vein. Spleen cells were harvested 5–6 h post-injection, stained with FITCconjugated anti-IL-4 and analyzed by flow cytometry. Immunization with anti-IgD was performed as described (19). Spleen cells were harvested 10 days post-injection, stimulated overnight in vitro with plate-bound anti-CD3, stained with FITC-conjugated anti-IL-4 and analyzed by flow cytometry.

	Results and discussion

Top Abstract Introduction Methods Results and discussion References

 
Generation of mIL-4 mice
The targeting construct used to generate mIL-4 mice is shown in Fig. 1. Briefly, the fourth exon of the IL-4 gene was modified by recombinant PCR to contain the connecting peptide, the transmembrane region and the first six amino acids of the cytoplasmic tail of the MHC class II A_ß^b gene. A cassette containing the neomycin-resistance gene was placed 150 nucleotides downstream of the polyadenylation signal of the IL-4 gene. The targeting construct was transfected into day 3 embryonic stem cells and homologous recombinants were identified by Southern analysis as described in Methods. One clone was used to generate chimeras that passed the mIL-4 allele through the germline. Heterozygous offspring were backcrossed six generations onto the BALB/c genetic background and then intercrossed to generate homozygous mIL-4 mice (Fig. 1).

Detection of IL-4-producing T_h2 cells
To determine if mIL-4 can be detected on the cell surface of differentiated CD4⁺ T_h2 cells, we used an in vitro differentiation protocol to enrich for this population. Spleen cells from wild-type and mIL-4 mice were stimulated with plate-bound anti-CD3 in the presence of IL-4 and anti-IL-12 for 7 days. Cells were then re-stimulated with plate-bound anti-CD3 for 24 h and analyzed by flow cytometry for the expression of mIL-4 using 11B11 mAb. mIL-4⁺ cells could not be detected from cultures of differentiated wild-type cells (Fig. 2) nor from cultures of unstimulated spleen cells irrespective of their genotype (data not shown). In contrast, ~20% of the in vitro differentiated CD4⁺ T cells from mIL-4 mice show a spectrum of positive staining with 11B11 mAb 24 h post secondary stimulation. These results demonstrate that mIL-4 expression can be used as a marker for the detection of IL-4-producing T_h2 cells. It should be noted that although nearly 75% of in vitro differentiated T_h2 cells from wild-type mice were positive for IL-4 when analyzed by intracellular cytokine staining (data not shown), this procedure does not allow for the recovery of viable cells. Thus, the detection of IL-4-producing cells by surface staining for mIL-4 represents a novel method for the recovery of viable IL-4-producing cells for further manipulation.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Detection of IL-4-producing Th2 cells from mIL-4 mice. Spleen cells from wild-type and mIL-4 mice were differentiated into Th2 cells in vitro. Twenty-four hours after re-stimulation with plate-bound anti-CD3, wild-type (bold solid line) and mIL-4 (solid line) cells were stained with FITC-conjugated anti-IL-4 or isotype-matched control (dotted line) antibody and analyzed by flow cytometry.

 
Detection of IL-4-producing peritoneal mast cells
Mast cells can secrete IL-4 following either cross-linking of FcR or stimulation with ionomycin (21). To determine if IL-4-secreting mast cells can be detected in mIL-4 mice, peritoneal mast cells from wild-type and mIL-4 mice were generated as described in Methods. Microscopic examination revealed that >50% of the peritoneal cells prepared by this method were large granule-containing cells having a morphology consistent with being mast cells. When analyzed by flow cytometry using 11B11 mAb, ~1–2% of this mast cell enriched population from mIL-4 mice stained positive at baseline (Fig. 3). Strikingly, >7% of the cells became positive for mIL-4 expression after only 6 h of stimulation with ionomycin. Flow cytometric analysis of ionomycin-activated cells from mIL-4 mice with an IL-2-specific antibody, or of ionomycin-activated cells from wild-type mice with 11B11 mAb, failed to reveal specific staining. Thus, these data demonstrate that IL-4-producing mast cells from mIL-4 mice can be detected by flow cytometry using 11B11 mAb.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Detection of IL-4-producing mast cells from mIL-4 mice. Enriched peritoneal mast cells from wild-type and mIL-4 mice were either stimulated with ionomycin or left unstimulated for 6 h. Cells were then stained with FITC-conjugated anti-IL-4, or FITC-conjugated anti-IL-2 as control, and analyzed by flow cytometry.

 
Detection of IL-4-producing NK T cells
NK T cells represent a small percentage of splenic lymphoid cells (~1%) but secrete large amounts of IL-4 upon ligation of their TCR by anti-CD3 in vivo (14). These cells are most easily identified by their cell surface expression of both CD4 and NK1.1. Since the mIL-4 mice were on a BALB/c genetic background and this strain does not express the NK1.1 marker, we mated mIL-4 mice to C57BL/6 mice to generate (BALB/cxC57BL/6)F₁ animals which expressed the NK1.1 marker and were heterozygous for the mIL-4 allele. These F₁ mIL-4 mice and wild-type C57BL/6 controls were injected with anti-CD3 i.v. When analyzed by flow cytometry 6 h post-injection with anti-CD3, ~1% of total spleen cells obtained from F₁ mIL-4 mice stained positive with 11B11 mAb (Fig. 4A). Consistent with previous reports documenting the phenotype of splenic IL-4-secreting NK T cells (22), these mIL-4⁺ cells were CD44^high, Mel14^low (Fig. 4B). Interestingly, none of the cells expressing high levels of NK1.1 stained positive for mIL-4, but rather mIL-4⁺ cells were equally divided into NK1.1^low and NK1.1^– subpopulations. This observation may be related to the recent demonstration that in vitro, expression of the NK1.1 marker is down-regulated on NK T cells following activation (23). Alternatively, at least some of the mIL-4⁺ NK1.1^– cells may represent memory T_h2 cells. Nevertheless, these results demonstrate that mIL-4 expression can be used as a marker for IL-4-producing NK T cells.

View larger version (85K): [in this window] [in a new window]   Fig. 4. Detection of IL-4-producing NK T cells from mIL-4 mice and expression of phenotypic markers. Spleen cells from wild-type C57BL/6 and (BALB/cxC57BL/6)F1 mIL-4 mice were isolated 6 h after i.v. injection with anti-CD3 or PBS and analyzed by flow cytometry using antibodies to the indicated cell surface markers.

 
In addition to their role in anti-CD3-induced IL-4 production, NK T cells are also thought to be the predominant producers of IL-4 following immunization with the polyclonal stimulus anti-IgD (24). CD1-deficient mice lack the major population of NK T cells and do not acutely produce IL-4 following in vivo stimulation with anti-CD3 (25–27). Nevertheless, these mice generate normal IgE antibody responses following administration of anti-IgD. These observations suggest that separate subpopulations of IL-4-secreting NK T cells, as defined by their ability to respond to different activating stimuli such as anti-CD3 or anti-IgD, may exist. To test this hypothesis, wild-type and mIL-4 mice were immunized with anti-IgD. Spleen cells were harvested 10 days after immunization and re-stimulated with plate-bound anti-CD3 in vitro for 24 h. Similar to that seen following injection of anti-CD3 in vivo, flow cytometric analysis of spleen cells from anti-IgD immunized mIL-4 mice demonstrated that ~1% of the cells were mIL-4⁺ and, of these, almost all were CD3⁺ CD4⁺ CD44^high Mel14^low (data not shown). Since, mIL-4 mice on the BALB/c genetic background do not express the NK1.1 marker, we examined the expression of the pan-NK cell marker DX5 on mIL-4⁺ cells. Approximately 30% of the mIL-4⁺ spleen cells derived from anti-IgD injected mIL-4 mice were positive for the DX5 marker (Fig. 5). In contrast, almost none of the mIL-4⁺ spleen cells derived from anti-CD3-injected mIL-4 mice expressed the DX5 marker (Fig. 4B and 5). Thus, these data suggest that there are at least two populations of IL-4-producing NK T cells; those that are DX5^– and stimulated in response to anti-CD3, and those that are DX5⁺ and activated in response to anti-IgD. While it is possible that the IL-4-producing DX5^– NK T cells seen in anti-IgD-injected mice are the same population of IL-4-producing NK1.1^low/– cells seen in anti-CD3-injected mice, the (BALB/cxC57BL/6) F₁ mIL-4 mice did not respond well to anti-IgD injection, thus preventing a direct test of this hypothesis. Nevertheless, these results demonstrate that IL-4-producing NK T cells are a heterogeneous population, and may explain the differences seen in the ability of CD1-deficient mice to mount acute versus chronic IL-4 responses to immunization with anti-CD3 and anti-IgD respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 5. DX5 expression on IL-4-producing NK T cells from anti-CD3 and anti-IgD Immunized mIL-4 mice. Spleen cells from wild-type and mIL-4 mice were either isolated 6 h after i.v. injection with anti-CD3 or harvested 10 days post-immunization with anti-IgD and stimulated overnight in vitro with plate-bound anti-CD3, and analyzed by flow cytometry for the expression of mIL-4 and DX5.

 
In this report, we have shown that multiple IL-4-secreting cell types from mIL-4 mice, including T_h2 cells, mast cells and NK T cells, could be detected by flow cytometry using an IL-4-specific mAb. Since the expression of the mIL-4 allele is driven by the endogenous IL-4 promoter in the context of its normal location in the genome, the transcription of this gene is regulated in a manner that is both quantitatively and qualitatively identical to the wild-type allele. Thus, knock-in of the IL-4 locus represents the most physiological approach to mark, track and/or sort IL-4-producing cells given the lack of a natural marker. mIL-4 mice will allow the easy identification and purification of IL-4-producing cells for the study of their roles in animal models of disease.

	Acknowledgments

 
This work was supported by a gift from the Mathers Foundation (L. H. G.), and National Institutes of Health grants AI/AG-87833 (L. H. G.) and AI-40171 (M. J. G.). I.-C. H. is an Investigator of the Arthritis Foundation. M. H. K. is a Special Fellow and M. J. G. is a Scholar of the Leukemia Society of America.

	Abbreviations

 

CTL	cytotoxic T lymphocyte
mIL-4	membrane-bound IL-4

	Notes

 
Transmitting editor: A. Singer

Received 25 June 1998, accepted 22 October 1998.

	References

Top Abstract Introduction Methods Results and discussion References

 

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