PE-conjugated streptavidin, PE-conjugated anti-human CD4, PE-conjugated anti-human γc, and Alexa Fluor®647 conjugated anti-human IL-7Rα antibodies were from BD Biosciences Pharmingen (San Jose, CA, USA). Anti-phosphotyrosine antibodies (HRP-conjugated) were from Millipore (Billerica, MA, USA) and anti-Stat5a antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant mouse IL-3, recombinant human IL-7, recombinant human TSLP and biotinylated anti-human TSLPR antibodies were from R&D Systems (Minneapolis, MN). RPMI 1640, fetal bovine serum (FBS), L-glutamine, and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). QuikChange XL-II mutagenesis kit was from Stratagene (La Jolla, CA, USA). Sequencing services were performed by the DNA and Peptide Synthesis and Sequencing facility at Johns Hopkins University School of Medicine using dye terminator chemistry. All other reagents used in this study were from Fisher Scientific (Pittsburgh, PA, USA).
The interleukin-3 (IL-3)-dependent pre B cell line, Ba/F3, was grown in RPMI 1640 supplemented with FBS, L-glutamine, penicillin, streptomycin, and mouse recombinant IL-3 (10 ng/ml). Stably transfected Ba/F3 cell lines were grown in RPMI 1640 supplemented with heat-inactivated FBS, L-glutamine, penicillin, streptomycin and mouse recombinant IL-3 (10 ng/ml). Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco modified essential medium, high glucose, supplemented with FBS, L-glutamine, penicillin, and streptomycin. Cell lines were maintained in the exponential growth phase unless indicated otherwise.
Plasmids and expression vectors
The human TSLPR was obtained from Dr. James Ihle [19
]. The human IL-7Rα was subcloned into a bicistronic retrovirus vector pMX-IRES-GFP [20
] that expresses GFP while the human TSLPR and γc
were subcloned into a bicistronic retrovirus vector pMX-IRES-hCD4 [20
] that expresses the human CD4 antigen. The human IL-7Rα, γc
and TSLPR mutants were generated by site-directed mutagenesis and confirmed by sequencing.
Generation of stable cell lines using retroviruses
HEK293T cells were transfected with the retroviral vector constructs (12 μg) together with the helper virus pCL-ECO (12 μg) (Imegenex, San Diego, CA, USA) by using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the supernatants were harvested and filtered through a 0.45-μm filter. Ba/F3 cells were infected with the pairs of the human IL-7Rα in pMX-IRES-GFP and the human TSLPR or γc in pMX-IRES-hCD4. 48 hours after infection, Ba/F3 cells were stained by PE-conjugated anti-human CD4 antibodies and then sorted for GFP and the human CD4 expression.
Receptors cell surface expression
After sorting, exponentially growing Ba/F3 cells expressing the pair of the wild type/mutated TSLP receptor complex were washed twice, stained with biotinylated anti-human TSLPR antibodies followed by PE-conjugated streptavidin and Alexa Fluor®647 conjugated anti-human IL-7Rα and analyzed by flow cytometry. Similarly, exponentially growing Ba/F3 cells expressing the pair of the wild type/mutated IL-7 receptor complex were washed twice, stained with PE-conjugated anti-human γc, and Alexa Fluor®647 conjugated anti-human IL-7Rα antibodies and analyzed by flow cytometry.
Immunoprecipitation and Western blotting
Exponentially growing Ba/F3 cells expressing different combinations of receptors were washed three times with RPMI 1640, deprived of IL-3 for 16 h, and then left untreated or stimulated with recombinant human TSLP or mouse IL-3 for 10 minutes at 37°C. Cells were lyzed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM sodium orthovanadate in the presence of protease inhibitors) followed by centrifugation. The supernatant was subjected to immunoprecipitation using anti-Stat5a antibodies. After washing, the immunoprecipitates were resolved by SDS-PAGE and assayed by Western blotting with anti-phosphotyrosine antibodies (4G10) followed by reprobing with anti-Stat5a antibodies.
The growth of cells expressing different combination of receptors was examined in 3 independent sets of experiments. In each experiment, exponentially growing Ba/F3 cells (in triplicate) expressing different combinations of receptors were washed three times with RPMI 1640, deprived of IL-3 for 16 h, and then stimulated with recombinant human TSLP or IL-7. Living cells were counted using a Beckman Coulter Z1 (Beckman Coulter, Fullerton, CA, USA). The results (except where indicated) are expressed as increase in cell number (stimulation [n-fold]) as compared to the number of cells plated on day 0. The mean and S.E.M were calculated for each independent experiment. The results from the three independent experiments were similar in each case and one representative experiment is shown in the figures.
Protein sequence alignments
The protein sequence alignments were performed using ClustalX version 2.0.10 using the BLOSUM62 matrix.
Data were expressed as mean ± SEM. Differences were examined by Student's t-test between two groups. p < 0.05 was considered significant.