Lymphocytes from Mice (Interleukins/T-Cell Development/Growth/Differentiation Factors) PASCHALIS SIDERAS*T, KEIKO FUNA*, ILANA ZALCBERG-QUINTANA*, KLEANTIS G

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Proc. Natl. Acad. Sci. USA Vol. 85, pp. 218-221, January 1988 Immunology Analysis by in situ hybridization of cells expressing mRNA for interleukin 4 in the developing thymus and in peripheral lymphocytes from mice (interleukins/T-cell development/growth/differentiation factors) PASCHALIS SIDERAS*t, KEIKO FUNA*, ILANA ZALCBERG-QUINTANA*, KLEANTIS G. XANTHOPOULOS§, PAWEL KISIELOW1, AND RONALD PALACIOS¶ Departments of *Immunology and §Microbiology, University of Stockholm, Stockholm, Sweden; tThe Ludwig Institute for Cancer Research, Uppsala Branch, University Hospital, Uppsala, Sweden; and 1The Basel Institute for Immunology, Basel, Switzerland Communicated by Niels K. Jerne, September 21, 1987

ABSTRACT We have made use of RNARNA in situ capacity to produce IL-4 (16). The latter clones possess a hybridization to study the presence of cells producing mRNA phenotype of helper T cells-i.e, they are Thy-1+, L3T4', for interleukin 4 (IL-4) in the developing thymus, spleen, and Lyt-2-, and express the receptor for IL-2 (P.S., unpublished T-cell line 2.19. Approximately 1 of 300-4 spleen cells results)-and have been proving useful for the isolation of expressed detectable IL-4 mRNA 24 hr after their stimulation cDNAs encoding IL-4 (1) and other lymphokines (17). by the lectin concanavalin A. Spleen cells were also induced to Both the interference of unrelated cytokines in the assay express mRNA for IL-4 by stimulation with alloantigens. systems used to detect IL-4 activity and the outgrowth of Splenocytes producing mRNA for IL-4 were detected 4 hr after unrelated cells have made any attempt to determine the stimulation by concanavalin A; the response peaked at -24 hr frequency of IL-4-secreting cells in normal tissues unsuc- and was undetectable by 72 hr. Cyclosporin A inhibited the cessful. Therefore, we opted for using RNA-RNA in situ synthesis of IL-4 mRNA in the T-cell line 2.19, which had been hybridization to study the presence of cells expressing induced by concanavalin A. Approximately 1 of 10 fetal mRNA for IL-4 in the developing thymus and in peripheral thymocytes at day 14 of gestation expressed mRNA for IL-4 lymphoid organs from adult mice. Here we report the results after their stimulation by phorbol 12-myristate 13-acetate and of such experiments. ionomycin. Both the frequency of fetal thymocytes expressing IL-4 mRNA and the amount of mRNA for IL4 synthesized per MATERIALS AND cell sharply decreased at day 16 of gestation, and <1 of 1800 METHODS fetal thymocytes at day 18 of gestation expressed detectable Reagents. RPMI 1640 medium was from GIBCO, conca- IL-4 mRNA. Our results define the relative frequency of cells navalin A (Con A) was from Pharmacia, Gemini SP6/T7 capable of expressing IL-4 mRNA after stimulation in vitro in vectors and the Riboprobe transcription kit were from the spleen and in the developing thymus. The data strongly Promega Biotec (Madison, WI), uridine 5'-[a-(35S)thio]tri- argue for an important role of IL-4 in growth and differenti- phosphate (UTP[35S]) was from New England Nuclear, ation of lymphoid cells, notably during T-cell development phorbol myristate acetate (PMA) was from Sigma, and within the thymus. ionomycin was from Calciochem. Mice. C3H/Tif, CBA/J, C57BL/6, and ATH 8- to 12-week- Recently, complementary DNA for the gene encoding the old mice were from the animal facility of the Department of glycoprotein interleukin 4 (IL-4) was isolated (1). IL-4, Immunology, University of Stockholm. Embryos from C57- previously called B cell-stimulatory factor 1 (2) or B-cell BL/6 mice bred at the Basel Institute for Immunology were differentiation factor for IgGi (3), is now known to possess obtained from timed matings; the day ofdetection ofa vaginal several biological activities. Thus, recombinant and highly plug was designated as day 0. purified IL-4 induces activated B lymphocytes to switch from Separation of Cells. Spleen cell suspensions were prepared IgM to IgG1 and IgE (1, 4-6), increases the level of Ia by standard procedures (5, 16). Thymocyte cell suspensions molecules on the cell membrane ofresting B lymphocytes (1, from fetal mice (days 14-18 of gestation) were prepared as 2, 7), supports proliferation of some interleukin 2 (IL-2)- described (18). The T-cell line 2.19 has been described and dependent cell lines (8-10) as well as of mast cell lines (8, 9), was maintained in culture as detailed elsewhere (16). promotes growth of early lymphoid precursors from bone Stimulation of Cells. The IL-4-producer T-cell line 2.19, marrow (11), and induces both growth and differentiation of suspended in culture medium (RPMI 1640 medium supple- normal intrathymic T-cell precursors from fetal mice in vitro mented with 2 mM L-glutamine, 2% penicillin, 2% strepto- (12). mycin, 5% fetal calf serum, and 50 at Although receptors specific for IL-4 on different types of IAM 2-mercaptoethanol) target cells have been demonstrated (13, 14), the mecha- 0.5 x 106 cells per ml, was stimulated with Con A (3 ug/ml) nism(s) by which IL-4 exerts its broad spectrum of activities at 370C for 6 and 24 hr. Freshly isolated spleen cells from is still unknown. Likewise, the frequency, tissue distribution, several strains of mice were cultured (106 cells per ml of and ontogeny of the cells capable of producing IL-4 are not culture medium) in the presence of Con A (3 ,ug/ml) at 370C known. All of the information available concerning the for 4, 12, 24, 48, and 72 hr. In some experiments, CBA/J production of IL-4 comes from studies performed with tumor spleen cells (2.5 x 107 per ml) were stimulated with x-ray- T-cell lines (15), with T-cell hybrids (3) and, recently, with irradiated allogeneic spleen cells (2.5 x 107 per ml) from antigen-specific T-cell clones that were selected for their Abbreviations: IL-2, IL-3, and IL-4, interleukins 2, 3, and 4; Con A, concanavalin A; PMA, phorbol 12-myristate 13-acetate. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Medical Chemistry, Kyoto University Faculty of Medicine, Sakyo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Ku, Kyoto 606, Japan. 218 Immunology: Sideras et al. Proc. Natl. Acad. Sci. USA 85 (1988) 219 C57BL/6 or BALB/c mice at 370C for 72 hr. The cells from In Situ Hybridization. Fixed slides prepared as described the different cultures were harvested, the dead cells were above were rinsed twice in 2x SSC (2x SSC = 0.3 M removed by centrifugation over density gradients (Lympho- NaCI/0.03 M sodium citrate) and then acetylated at pH 8 with pred), and the viable cells were used for cytocentrifuge 0.25% acetic anhydride for 10 min. After this, the slides were preparations. Fetal thymocytes (2.0 x 106 cells per ml of rinsed in 2x SSC and phosphate-buffered saline and were culture medium) were stimulated with PMA (10 ng/ml) and immersed in 0.1 M Tris.HCI/0.1 M glycine, pH 7.0 for 30 min ionomycin (500 ng/ml) at 370C for 4-6 hr. The cells were before hybridization. harvested, washed, and used for cytocentrifuge preparations. Twenty microliters of probe mixture [15 u1d of formamide Cytocentrifuge Preparations. Glass slides were immersed in containing 20% dextran sulfate, 2 1,l ofsheared herring sperm 70% ethanol and air-dried. The cells suspended in balanced DNA (10 mg/ml), 2 ,ul of bovine serum albumin (nuclease salt solution containing 10%o fetal calf serum were loaded on free; 20 mg/ml), and 1 ,ul of labeled RNA (106 cpm/,ul)] was glass slides (105 cells per slide) with the aid of a cytocentri- loaded on each slide, and hybridization was performed at fuge. The cell smears were fixed in paraformaldehyde (4% 50°C for 3 hr. The slides were washed three times with 50% diluted in phosphate-buffered saline) for 1 min, were trans- formamide/2x SSC at 52°C, rinsed in 2x SSC, and treated ferred into a solution of 70% ethanol, and subsequently were with 30 ,ul of RNase solution for 30 min at 37°C (100 ,ug of stored at +40C until used. RNase A plus 1 ,tg of RNase T1 per ml). The slides were Preparation of the IL4 Probe. The Rsa I-Rsa I fragment rinsed twice in 2x SSC and once in 50% formamide/2 X SSC from the cDNA clone pSP6KmIL4-293 (1), containing all of at 52°C, rinsed again three times in 2x SSC, dehydrated the coding sequence for IL-4, was subcloned into the gemini SP6/T7 vectors. The pUC18 vector containing the Rsa I-Rsa successively in 70%, 80%o, and 90%o ethanol, air-dried, and I fragment was kindly provided by T. Honjo (Kyoto Univer- finally autoradiographed. Kodak nuclear track emulsion sity, Kyoto, Japan). Linearized plasmids were used as (NTB-2, Eastman Kodak) was melted at 43°C and diluted templates for the synthesis in vitro of radioactive RNA with an equal volume of water. The slides were dipped into probes complementary to the cellular IL-4 mRNA. RNA was the emulsion and allowed to solidify horizontally at room also transcribed from the opposite direction (sense) and used temperature for 3-4 hr. The emulsion-coated slides were kept as a negative control. Approximately 2 x 108 cpm were at +4°C for 3-5 days for exposure. The slides were developed incorporated into RNA per tug of RNA template. The size of in Kodak developer 19 at 15°C for 5 min. After a rinse in tap the full-length probe mRNA was =400 nucleotides. Nonra- water, the fixation was carried out in Kodak fixer for 5 min, dioactive RNA was also synthesized in the sense orientation and the slides were washed with water for 30 min. The slides and used to compete with the binding of the radioactive were stained with 10o Giemsa stain for 2.5 min, washed probes to the cellular mRNA. twice with tap water, and air-dried. We considered as

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FIG. 1. Detection by in situ hybridization of IL-4 mRNA in the T-cell clone 2.19 and splenocytes. Photomicrographs of Con A-stimulated 2.19 cells (A) or CBA/J spleen cells stimulated by allogeneic C57BL/6 irradiated spleen cells (B) were hybridized with an RNA probe complementary to the cellular IL-4 mRNA. 2.19 cells stimulated for 6 hr with Con A were treated with RNase before hybridization with the RNA probe complementary to the IL-4 mRNA (C). 2.19 cells stimulated by Con A for 6 hr were hybridized with the IL-4 mRNA probe in the sense orientation (D). (x 1000.) 220 Immunology: Sideras et al. Proc. Natl. Acad. Sci. USA 85 (1988) Table 1. Cyclosporin A inhibits the expression of mRNA for IL-4 in the 2.19 T-cell clone induced by Con A 2.19 cells expressing IL-4 mRNA after Con A stimulation for 2, 4, 12, and 24 hr, % Cyclosporin A 2 hr 4 hr 12 hr 24 hr Without 58 (32 ± 23) 68 (76 ± 48) 65 (80 ± 58) 7 (34 ± 2) With <0.01 (11 ± 2) <0.01 (15 ± 6) ND ND The 2.19 cells were stimulated with Con A in the presence or absence of cyclosporin A at 370C for various periods (2 4, 12, and 24 hr). The presence of 2.19 cells expressing mRNA for IL-4 was assessed by RNA-RNA in situ hybridization. Numbers in parentheses are the mean ± SEM of grains counted on the positive cells. Unstimulated 2.19 cells contained <1% cells expressing detectable IL-4 mRNA. ND, not detectable. positive cells (cells expressing IL-4 mRNA) those cells that presence of cells in the spleen from several mouse strains had >10 grains per cell. expressing IL-4 mRNA following their stimulation either by the polyclonal T-cell activator Con A or by alloantigens. The RESULTS AND DISCUSSION results (Table 2) show that about 1 of 300-400 spleen cells stimulated by Con A for 48 hr expressed IL-4 mRNA as The T-cell line 2.19, known to produce IL-4 (5, 16) and the assessed by in situ RNA-RNA hybridization. Spleen cells original source from which cDNA for the gene encoding IL-4 expressing IL-4 mRNA after activation by alloantigens were was isolated (1), was initially used to optimize the conditions clearly observed, but at much lower frequency than that for the detection of mRNA for IL-4 by in situ hybridization. found in Con A-stimulated spleen cells (Fig. 1B; and data not Fig. 1 depicts the results obtained with 2.19 T cells that were shown). Kinetic studies of the appearance of spleen cells stimulated for 6 hr (Fig. 1A) with the lectin Con A. Approx- expressing IL-4 mRNA after stimulation with Con A show imately 85% of the 2.19 cells stimulated with Con A for 6 hr that cells expressing mRNA for IL-4 could be detected 4 hr hybridized with the radiolabeled DNA probe complementary after initiation of the cultures. By 12 hr of culture, both the to the cellular IL-4 mRNA. When 2.19 cells were stimulated number of cells producing IL-4 mRNA and the amount of by Con A for 24 hr, -z30% of the cells still expressed IL-4 IL-4 mRNA expressed by each cell (asjudged by the number mRNA. of grains counted per cell) have significantly increased, Next, we checked the specificity of the in situ RNARNA reaching maximum levels 24 hr after initiation ofthe cultures. hybridization procedure by incubating cytocentrifuge prep- The number of cells synthesizing IL-4 mRNA slowly in- arations of 6-hr Con A-stimulated 2.19 T cells with RNA creased up to 48 hr of culture, but the amount of mRNA for probes in the sense orientation (i.e., same polarity as the IL-4 per cell decreased, and, by 72 hr of culture, no cells cellular mRNA) and by hybridizing RNase-treated cytocen- expressing IL-4 mRNA were detected (Table 2). It is worth trifuge preparations of 2.19 cells with RNA probes in the noting that even at the peak ofthe response, the frequency of antisense orientation (i.e., complementary to cellular mRNA). spleen cells expressing IL-4 mRNA was not higher than 1 in The results show that no hybridization could be observed with =300 viable cells and that the actual increase in the number either the probe in the wrong orientation (Fig. 1D) or when ofcells producing IL-4 mRNA was not marked (i.e., between the slides were pretreated with RNase (Fig. 1C). Further- 12 and 48 hr after activation, the number of cells producing more, cold in vitro synthesized IL-4 mRNA in the sense IL-4 mRNA only increased 2-fold). These findings may orientation, included during the hybridization procedure, indicate the capacity ofnonblastoid spleen cells to synthesize inhibited the binding of the complementary mRNA probe to high levels of mRNA for IL-4 (in Table 2, see the column the cellular IL-4 mRNA (not shown). designated "% blasts") and the limited proliferative activity We also studied the effect of cyclosporin A, a drug known ofthe cell populations expressing IL-4 mRNA following their to inhibit the transcription of genes coding for the growth activation by Con A. factors IL-2 and interleukin 3 (IL-3) (19), on the expression We recently have described that recombinant IL-4 pro- of IL-4 mRNA by 2.19 T cells stimulated with Con A. The motes the growth of about one of four intrathymic T-cell results (Table 1) show that treatment of the 2.19 T cells with precursors from fetal mice (days 14-15 of gestation) (12). It cyclosporin A inhibited their capacity to express IL-4 mRNA was considered important to determine whether fetal thymo- after Con A stimulation. The viability of the 2.19 T cells cytes can express mRNA for IL-4. Thus, fetal thymocytes at exposed to the drug was similar to that of nontreated Con various ages of gestation (days 14, 15, 16, 17, and 18) were A-stimulated 2.19 T-cell cultures. stimulated with the combination of PMA and the calcium Having defined the conditions and checked the specificity ionophore ionomycin for 4-6 hr, and the presence of cells of the in situ hybridization procedure, we next studied the expressing IL-4 mRNA was assessed by in situ RNA-RNA Table 2. Kinetics of spleen cells expressing mRNA for IL-4 induced by Con A Mouse Time after Con A Positive cells strain stimulation, hr No. per 106 cells Grains per cell % blasts Frequency ATH 4 2300 64 ± 36 0 1/540 12 3300 128 ± 38 2 1/380 24 4000 260 ± 90 65 1/250 48 5600 170 ± 64 85 1/380 72 <1/10,000 C57BL/6 4 1800 56 ± 18 1 1/660 12 2000 92 18 2 1/600 24 3100 154 46 40 1/450 48 4200 94 54 70 1/500 72 <1/10,000 A similar phenomenon was observed when using spleen cells from C3H/TiF and CBA/J mice. Immunology: Sideras et al. Proc. Natl. Acad. Sci. USA 85 (1988) 221

A Table 3. Activated fetal thymocytes express mRNA for IL-4 Relative frequency Day of of fetal thymocytes Grains per cell, no. gestation expressing IL-4 mRNA Mean SD SEM 14 1/10 43.7 25.6 4.20 15 1/28 39.0 22.0 6.00 16 1/100 26.9 16.5 3.38 17 1/800 20.9 12.8 3.57 18 <1/1800 15.0 7.07 5.00 Fetal thymocytes were stimulated with PMA/ionomycin at 370C for 4-6 hr, and the cells expressing mRNA for IL-4 were assessed by RNA-RNA in situ hybridization.

V~~~~'. levels of IL-4 mRNA expressed per cell decrease as they r ._ differentiate within the thymus. B s We are grateful to Dr. T. Honjo for the pUC18 plasmid containing the IL-4 Rsa I-Rsa I fragment, Drs. G. Mdller and T. Honjo for critical reading of the manuscript, and Ms. C. Plattner for the excellent and patient preparation of the manuscript. This work was supported by grants from the Karolinska Institutet, the Swedish Cancer Society, and The Swedish Medical Research Council. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Ltd. Co., Basel, Switzerland.

1. Noma, Y., Sideras, P., Naito, T., Bergsted-Lindquist, S., Azuma, S., Severinson, E., Tanabe, T., Kinashi, T., Matsuda, F., Yaoita, Y. & Honjo, T. (1986) Nature (London) 319, 640-646. 2. Rabin, E., Ohara, J. & Paul, W. E. (1985) Proc. Nati. Acad. Sci. USA 82, 2953-2958. 3. Isakson, P., Pure, E., Vitetta, E. & Krammer, P. (1982) J. Exp. FIG. 2. Fetal thymocytes at day 14 (A) and day 15 (B) ofgestation Med. 155, 734-741. expressing mRNA for IL-4 induced by PMA/ionomycin stimulation 4. Vitetta, E., Ohara, J., Myers, C., Layton, J., Krammer, P. and assessed by RNA-RNA in situ hybridization. (x 1000.) Paul, W. E. (1985) J. Exp. Med. 162, 1726-1731. 5. Sideras, P., Bergsted-Lindquist, S. & Severinson, W. (1985) hybridization. Previously, we have shown that PMA/ Eur. J. Immunol. 15, 593-598. ionomycin are effective inducers of immature cells to pro- 6. Coffman, R., Ohara, J., Bond, J., Carty, J., Zlotnick, A. & duce the growth factor IL-3 (20) as well as of immature Paul, W. E. (1986) J. Immunol. 136, 4538-4544. 7. Noelle, R., Krammer, P., Ohara, J., Uhr, J. & Vitetta, E. thymocytes for responding to IL-2 (21). Fig. 2 illustrates 14- (1984) Proc. Nati. Acad. Sci. USA 81, 6149-6153. and 15-day fetal thymocytes that express mRNA for IL-4 8. Lee, F., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., after stimulation by PMA/ionomycin. Some of these cells Coffman, R., Mosmann, T., Rennick, D., Roehm, N., Smith, express high levels of IL-4 mRNA as judged by the number C., Zlotnik, A. and Arai, K.-i. (1986) Proc. Nati. Acad. Sci. of grains per cell (Table 3). Remarkably, fetal thymocytes at USA 83, 2061-2065. day 14 ofgestation contained both the highest number ofcells 9. Grabstein, K., Eisenman, J., Mochizuki, D., Shanebeck, K., producing IL-4 mRNA and the cells expressing the highest Conlon, P., Hopp, T., March, C. & Gillis, S. (1986) J. Exp. Med. 163, 1405-1414. levels of mRNA for IL-4 (Table 3). The frequency of fetal 10. Fernandez-Botran, R., Krammer, P., Diamanstein, T., Uhr, J. thymocytes expressing IL-4 mRNA sharply decreased at day & Vitetta, E. (1986) J. Exp. Med. 164, 580-593. 16 of gestation, and very few IL-4 mRNA-producing cells 11. Sideras, P. & Palacios, R. (1987) Eur. J. Immunol. 17, 217-221. were observed in 18-day fetal thymocytes (Table 3). The data 12. Palacios, R., Sideras, P. & von Boehmer, H. (1987) EMBO J. show that 1 ofabout 10 fetal thymocytes at day 14 ofgestation 6, 91-95. can express mRNA for IL-4 after their activation by PMA/ 13. O'Hara, J. & Paul, W. E. (1987) Nature (London) 325, 537-540. and that the number 14. Park, L., Friend, D., Grabstein, K. & Urdal, D. (1987) Proc. ionomycin offetal thymocytes with such Nati. Acad. Sci. USA 84, 1669-1673. potential decreases as they differentiate within the thymus. 15. Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, The present results provide strong support for the proposition K., Hamaoka, K. & Paul, W. E. (1982) J. Exp. Med. 155, that IL-4 may play an essential role in the early stages of 914-923. T-cell development within the thymus, promoting growth and 16. Sideras, P., Bergsted-Lindquist, S., MacDonald, R. & Sever- differentiation of the T-cell progenitors (12). inson, E. (1985) Eur. J. Immunol. 15, 586-593. In summary, in situ RNARNA 17. Kinashi, T., Harada, N., Severinson, E., Tanabe, T., Sideras, by using hybridization for P., Konishi, M., Azuma, C., Tominaga, A., Bergsted-Lind- the detection at the single-cell level of expression of IL-4 quist, S., Takahashi, M., Matsuda, F., Yaoita, Y., Takatsu, K. mRNA, we have provided here evidence that: (i0 about 1 of & Honjo, T. (1986) Nature (London) 324, 70-73. =300 spleen cells may express mRNA for IL-4 after stimu- 18. Kisielow, P., Leiserson, W. & von Boehmer, H. (1984) J. lation by Con A; (ii) that cyclosporin A inhibits the expres- Immunol. 133, 1117-1123. sion of IL-4 mRNA by the T-cell line 2.19 induced by Con A; 19. Herold, K., Lancki, D., Moldwin, L. & Fitch, F. (1986) J. Immunol. 136, 1315-1321. and (iii) that about 1 of 10 fetal thymocytes at day 14 of 20. Palacios, R. & Leu, T. (1986) Cell. Immunol. 100, 532-540. gestation expresses IL-4 mRNA after proper stimulation in 21. Palacios, R. & von Boehmer, H. (1986) Eur. J. Immunol. 16, vitro and that both the number of fetal thymocytes and the 12-19.