Materials and Reagents

Absorbent cotton
1 ml syringes (Terumo, catalog number: SS-01T)
26 G needles (Terumo, catalog number: NN-2613S)
Micro slide glass (Matsunami, catalog number: S-2226)
Cell strainer (Thermo, catalog number: 08-771-2)
Cell lines
1. B300-19 cells
  IL-9R/B300-19 and EV/B300-19 cells: B300-19 cells (a mouse pre-B cell line), transduced with a retroviral vector containing Il9r cDNA (pMXs-IL9R-IRES-hNGFR) or an empty vector (EV; pMXs-IRES-hNGFR). The B300-19 cell line was provided by Dr. Takashi Nakayama. The retroviral vectors and B300-19 transfectants generated by us will be available upon request.
2. Plat-E cells
  The Plat-E cell line and an original pMXs vector were provided by Dr. Toshio Kitamura.
Lew/SSN rats (Female Lew/SSN rats were purchased from Sankyo Lab Service. Rats were immunized at 4 weeks of age)
Depilatory cream (general commercialized product)
Sodium pentobarbital (Kyoritsu Seiyaku, Somnopentyl Injection)
QIAGEN Plasmid Giga Kit (QIAGEN, catalog number: 12191)
Fugene HD (Promega, catalog number: E2311)
FITC-conjugated secondary antibodies [FITC-conjugated goat anti-rat IgG (H+L)] (SouthernBiotech, catalog number: 3050)
FITC-anti-IgG1 (BD Bioscience, catalog number: 562580)
PE-anti-CD138 (Biolegend, catalog number: 142503)
PE-anti-IgM (Thermo, catalog number: 12-5790-81)
PE-anti-IgD (Biolegend, catalog number: 405705)
PerCP/Cy5.5-anti-T and -B Cell activation antigen clone GL-7 (Biolegend, catalog number: 144609)
PE/Cy7-anti-CD38 (Thermo, catalog number: 25-0381-80)
APC/Cy7-anti-B220 (Biolegend, catalog number: 103223)
Biotin-NP₁₄-BSA (in house)
Brilliant Violet 421 Streptavidin (Biolegend, catalog number: 405226)
MEM medium (Thermo, catalog number: 11095080)
Hyaluronidase (Sigma, catalog number: H3631-30KU)
70% (v/v) Ethanol
Phosphate buffered saline (pH 7.4)
Tris
EDTA
Sodium azide
Hyaluronidase solution (see Recipes)
Tris-EDTA buffer (see Recipes)
FACS Buffer (see Recipes)
Plasmid DNA solution (see Recipes)

Equipment

Electroporator (BTX, Electro Square Porator ECM830, Figure 1A)
Copper cables with IC hook (Toyoshima, made-to-order or general commercialized product, length: more than one meter, Figure 1A-1B)
26 G two-stage needle (Toyoshima, made-to-order, Figure 1C-1D)
Gamma irradiation device (Atomic Energy of Canada Limited, Gammacell 40)
BD FACS Calibur
BD FACS CantoII

Figure 1. Details of the experimental equipment for DNA immunization. A. Electroporator. B. Electrodes and copper cables with IC hook. C. 1 ml syringe with a 26 G two-stage needle. D. 26 G two-stage needle.

Software

FlowJo (Tree Star, https://www.flowjo.com)

Procedure

Prepare and set up the electroporator (Figure 1A):
Voltage: 100 V
Pulse length: 50 ms
Pulse number: 6 (1 s interval)
Periodicity: 1 kHz.
Anesthetize rat with 24-36 mg/kg sodium pentobarbital. (Duration time: 30-60 min)
Depilate hindlimbs with depilatory cream (cream should stay for about 3 min) (Figure 2A).
Sterilize hindlimbs using absorbent cotton impregnated with 70% (v/v) ethanol.
Inject 50 μl of hyaluronidase solution into the quadriceps muscle using a 1 ml syringe with a 26 G two-stage needle (Figure 2B).
Leave to stand for 10 min.
Stab electrodes into the quadriceps muscle (Figures 1B and 2C).
Inject 30 μl of plasmid DNA solution into the same place where hyaluronidase solution pretreatment was done (Figure 2D).
Pulse by electroporator (100 V, 50 ms pulse length, 1 kHz, 6 times with each 1 s interval).
Repeat the same operation (Steps 3-9) to the other hindlimb (Figure 2A-2D).
Note: The required time of operation (Steps 3-10) is about 20 min.

Figure 2. Experimental procedure of DNA immunization using in vivo electroporation. A. Depilate hindlimbs. B. Inject hyaluronidase solution into quadriceps muscle and leave to stand for 10 min. C. Stab the electrodes into the quadriceps muscle. D. Inject plasmid DNA solution into the same place where hyaluronidase solution pretreatment was done. Subsequently, pulse by electroporator. Repeat the same operation (A-D) to the other hindlimb.
Repeat the same DNA-immunization protocol 1, 3, 4, 7 and 9 weeks after the primary immunization, respectively (Figure 3).

Figure 3. Overview of DNA immunization using rats. Scheme of DNA immunization using in vivo electroporation.
Irradiate (30 Gy) 1 x 10⁹ IL-9R/B300-19 cells in PBS (preferably on ice) using Gamma irradiation device.
Transfer 1 x 10⁹ irradiated IL-9R/B300-19 cells intraperitoneally 4-27 weeks after the final immunization.
Six or eleven weeks after the primary immunization, check sera of the immunized rats for anti-IL-9R IgG by flow cytometry using IL-9R/B300-19 cells. Detection with sera diluted by 1:10⁴ or more is desirable (Figure 4A).
In order to generate hybridomas that produce monoclonal antibodies, collect splenocytes from the immunized rats 3 days after the cell transfer, and fuse them with mouse myeloma cell line (e.g., SP2/0) by a conventional method (e.g., Köhler and Milstein, 1975).
Screen the hybridomas for secretion of IgG Ab that stains IL-9R/B300-19 cells (and does not stain EV/B300-19 cells) using flow cytometry (Figure 4B).

Figure 4. Titration of antisera and hybridoma screening. A. Titration of anti-IL9R antisera using flow cytometry. IL-9R/B300-19 cells were stained with sera, diluted (dil) as indicated, of a rat, immunized 6 weeks previously. B. An example of flow cytometric analysis in hybridoma screening. IL-9R/B300-19 or EV/B300-19 cells were stained with hybridoma supernatant. (A-B) Bound IgGs were revealed with FITC-conjugated anti-rat IgG Ab.
Stain the cells expressing IL-9R utilizing anti-mouse IL-9R (e.g., Figure 5) (Takatsuka et al., 2018).

Figure 5. Detection of IL-9R expression on memory B cells. Flow cytometry analysis of IL-9R expression using anti-IL-9R mAb, RZ-103, (right panel) on NP-specific IgG1⁺ memory B cells in the spleen (gating strategies are shown on the left) of Il9r^+/+ or Il9r^−/− mice immunized i.p. with a hapten NP conjugated with chicken gamma globulin (NP-CGG) in alum 16 weeks earlier.

Data analysis

Flow cytometry analysis: For analysis, collect > 5,000 events (Figure 4) or > 5,000,000 events (Figure 5) for each sample. Exclude dead cells and cellular debris from analysis. Define singlet cells in a lymphocyte gate by forward, and side scatters. These cells can be visualized by the fluorescently labeled antibodies. (e.g., Figure 5: the analysis of the Flow Cytometry assay is done via FlowJo.)

Notes

Retroviral transduction
1. Transfect pMXs-IL9R-IRES-hNGFR vectors into Plat-E cells by Fugene HD.
2. Two days after transfection, harvest the virus-containing supernatant and add to B300-19 cells.
Preparation of serum
1. Cut up rat’s tail using a surgical knife and collect the peripheral blood (50-100 μl).
2. Leave to stand the peripheral blood at 4 °C, overnight.
3. Centrifuge the peripheral blood at 2,500 x g, 4 °C for 10 min.
4. Collect the serum (20-40 μl) and store at -20 °C.
Preparation of Single-cell suspensions from spleens
1. Grind the spleen by frosted glass portion of the slide glass in the MEM medium.
2. Filter splenocytes through a cell strainer.
Serum titer check using flow cytometry
1. Dilute the serum with FACS Buffer and add to 5 x 10⁴ IL-9R/B300-19 cells.
2. Leave to stand on ice for 20 min.
3. Wash two times each with 200 μl of FACS Buffer.
4. Add to FITC-conjugated secondary antibodies.
5. Leave to stand on ice for 20 min.
6. Wash two times each with 200 μl of FACS Buffer.
7. Go to flow cytometry analysis (see Data analysis).
Hybridoma screening using flow cytometry
1. Collect culture supernatants (25-50 μl) of hybridomas and add to 5 x 10⁴ IL-9R/B300-19 cells.
2. Leave to stand on ice for 20 min.
3. Wash two times each with 200 μl of FACS Buffer.
4. Add to FITC-conjugated secondary antibodies.
5. Leave to stand on ice for 20 min.
6. Wash two times each with 200 μl of FACS Buffer.
7. Go to flow cytometry analysis (see Data analysis).

Recipes

Hyaluronidase solution
Prepare a 1 Unit/μl bovine hyaluronidase solution in phosphate buffered saline
Tris-EDTA buffer
10 mM Tris, pH 7.5
1 mM EDTA
FACS Buffer
PBS (-)
0.5 % BSA
2 mM EDTA
0.05 % sodium azide
Plasmid DNA solution
Prepare a 10 mg/ml DNA solution (in Tris-EDTA buffer) of a plasmid carrying a gene encoding IL-9R-GroEL fusion protein, driven by a CAG promoter, as previously described (Fujimoto et al., 2009)

Acknowledgments

We thank Y. Takatsuka for figure preparation; K. Sano for proofreading. This work was supported by the Japan Society for the Promotion of Science KAKENHI, Grant-in-Aid for Scientific Research (B) (16H05206) and Grant-in-Aid for Scientific Research on Innovative Areas (25111512).

Competing interests

All authors declare that they have no competing interests.

Ethics

Lew/SSN rats were maintained in an animal facility in Research Institute for Biomedical Sciences, Tokyo University of Science under specific pathogen-free conditions and were treated under protocols approved by the Animal Care and Use Committee of the Tokyo University of Science.

References

Aihara, H. and Miyazaki, J. (1998). Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16(9): 867-870.
Bauer, J. H., Liu, K. D., You, Y., Lai, S. Y. and Goldsmith, M. A. (1998). Heteromerization of the gammac chain with the interleukin-9 receptor alpha subunit leads to STAT activation and prevention of apoptosis. J Biol Chem 273(15): 9255-9260.
Davis, H. L., Whalen, R. G. and Demeneix, B. A. (1993). Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum Gene Ther 4(2): 151-159.
Fujimoto, A., Kosaka, N., Hasegawa, H., Suzuki, H., Sugano, S. and Chiba, J. (2012). Enhancement of antibody responses to native G protein-coupled receptors using E. coli GroEL as a molecular adjuvant in DNA immunization. J Immunol Methods 375(1-2): 243-251.
Fujimoto, A., Takatsuka, S., Ishida, I. and Chiba, J. (2009). Production of human antibodies to native cytokine receptors using the genetic immunization of KM mice. Hum Antibodies 18(3): 75-80.
Furutani, M., Hata, J., Shomura, Y., Itami, K., Yoshida, T., Izumoto, Y., Togi, A., Ideno, A., Yasunaga, T., Miki, K. and Maruyama, T. (2005). An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool. Protein Sci 14(2): 341-350.
Gariboldi, S., Palazzo, M., Zanobbio, L., Selleri, S., Sommariva, M., Sfondrini, L., Cavicchini, S., Balsari, A. and Rumio, C. (2008). Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of β-defensin 2 via TLR2 and TLR4. J Immunol 181(3): 2103-2110.
Gounni, A. S., Nutku, E., Koussih, L., Aris, F., Louahed, J., Levitt, R. C., Nicolaides, N. C. and Hamid, Q. (2000). IL-9 expression by human eosinophils: regulation by IL-1β and TNF-α. J Allergy Clin Immunol 106(3): 460-466.
Kohler, G. and Milstein, C. (2005). Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. J Immunol 174(5): 2453-2455.
Licona-Limon, P., Henao-Mejia, J., Temann, A. U., Gagliani, N., Licona-Limon, I., Ishigame, H., Hao, L., Herbert, D. R. and Flavell, R. A. (2013). Th9 cells drive host immunity against gastrointestinal worm infection. Immunity 39(4): 744-757.
McMahon, J. M., Signori, E., Wells, K. E., Fazio, V. M. and Wells, D. J. (2001). Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase-increased expression with reduced muscle damage. Gene Ther 8(16): 1264-1270.
Noelle, R. J. and Nowak, E. C. (2010). Cellular sources and immune functions of interleukin-9. Nat Rev Immunol 10(10): 683-687.
Renauld, J. C., Druez, C., Kermouni, A., Houssiau, F., Uyttenhove, C., Van Roost, E. and Van Snick, J. (1992). Expression cloning of the murine and human interleukin 9 receptor cDNAs. Proc Natl Acad Sci U S A 89(12): 5690-5694.
Scheibner, K. A., Lutz, M. A., Boodoo, S., Fenton, M. J., Powell, J. D. and Horton, M. R. (2006). Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J Immunol 177(2): 1272-1281.
Stassen, M., Arnold, M., Hultner, L., Muller, C., Neudorfl, C., Reineke, T. and Schmitt, E. (2000). Murine bone marrow-derived mast cells as potent producers of IL-9: costimulatory function of IL-10 and kit ligand in the presence of IL-1. J Immunol 164(11): 5549-5555.
Takatsuka, S., Sekiguchi, A., Tokunaga, M., Fujimoto, A. and Chiba, J. (2011). Generation of a panel of monoclonal antibodies against atypical chemokine receptor CCX-CKR by DNA immunization. J Pharmacol Toxicol Methods 63(3): 250-257.
Takatsuka, S., Yamada, H., Haniuda, K., Saruwatari, H., Ichihashi, M., Renauld, J. C. and Kitamura, D. (2018). IL-9 receptor signaling in memory B cells regulates humoral recall responses. Nat Immunol 19(9): 1025-1034.
Ulmer, J. B., Donnelly, J. J., Parker, S. E., Rhodes, G. H., Felgner, P. L., Dwarki, V. J., Gromkowski, S. H., Deck, R. R., DeWitt, C. M., Friedman, A. and et al. (1993). Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259(5102): 1745-1749.
Wilhelm, C., Hirota, K., Stieglitz, B., Van Snick, J., Tolaini, M., Lahl, K., Sparwasser, T., Helmby, H. and Stockinger, B. (2011). An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation. Nat Immunol 12(11): 1071-1077.
Wolff, J. A., Ludtke, J. J., Acsadi, G., Williams, P. and Jani, A. (1992). Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet 1(6): 363-369.
Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L. (1990). Direct gene transfer into mouse muscle in vivo. Science 247(4949 Pt 1): 1465-1468.
Yamazaki, T., Nagashima, M., Ninomiya, D., Arai, Y., Teshima, Y., Fujimoto, A., Ainai, A., Hasegawa, H. and Chiba, J. (2011). Passive immune-prophylaxis against influenza virus infection by the expression of neutralizing anti-hemagglutinin monoclonal antibodies from plasmids. Jpn J Infect Dis 64(1): 40-49.