Patients
Three Chinese patients with mutations in the DOCK8 gene were enrolled in the study. All patients were admitted to the Children’s Hospital of Chongqing Medical University. Diagnosis of the patients was as previously described [18]. Three age-matched subjects were enrolled as healthy controls (HCs). Informed consent to participate in the study was provided by the patients’ families, and the study was approved by the Medical Ethics Committee of Children’s Hospital of Chongqing Medical University.
Mice
DOCK8 KO mice were generated using the TALEN technique (Shanghai Biomodel Organism Science & Technology Development Co., Ltd). The first exon of DOCK8 was chosen for TALEN-induced mutagenesis; absence of a 45 bp sequence from the coding frame of exon 1 introduced a reading frame shift into the DOCK8 gene [19]. DOCK8 was genotyped by PCR using the following primer pair: sense, 5’- GGGGGATCCCCTGCGGCCGGCGACTCTGA-3’, and antisense, 5’- GGGGAATTCGAAGCGGGGAAGGCAATGATGACA-3’. PCR products amplified from F0 generation mouse tail tissue were purified, cloned, and sequenced to identify positive founder mice with the DOCK8 protein harboring the frame shift. Positive F0 generation mice were crossed with C57BL/6J mice and the genotype of the offspring was confirmed by PCR, cloning, and sequencing. CD4KO and CD45.1+ C57BL/6 mice were obtained from the Jackson Laboratory. C57BL/6 mice were purchased from the Laboratory Animal Center, Chongqing Medical University. All mice were aged 6 to 10 weeks at the time of the experiments and were housed in specific pathogen-free animal facilities. Data were obtained from three or more mice per group. All animal experiments were reviewed and approved by the Institutional Animal Care and Usage Committee of Children’s Hospital of Chongqing Medical University.
Flow cytometry and phosphorylation analyses
Heparinized blood was obtained from patients and from age-matched HC subjects. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation and cell numbers were counted in a hemocytometer. Flow cytometry was performed using a FACSCanto II cytometer (BD Biosciences, San Jose, Calif). Briefly, PBMCs were stained with anti-human CD19-APC, anti-human CD24-PE, anti-human CD27-BV450, anti-human CD38-PerCP-cy5.5, or anti-human IgD-BV510. Mononuclear cells isolated from the spleen of DOCK8 KO and WT mice were stained with the following antibodies: anti-CD19 FITC, anti-CD5 PE, and anti-CD1d APC (CD1dhiCD5+ B cells); anti-CD19 FITC, anti-CD23 PE, anti-CD21 APC, and anti-CD24 Percp-cy5.5 (T2-MZP cells) (all antibodies were from BioLegend, CA). Mononuclear cells isolated from spleen cells of chimera mice were stained with anti-CD45.1 BV510, CD45.2 FITC, anti-CD19 APC, and anti-IL-10 PE (BioLegend, CA). Mononuclear cells isolated from the spleen of CD4 KO mice were stained with anti-CD19 FITC or APC, anti-CD5 PE, anti-CD1d APC, anti-CD23 PE, anti-CD21 APC, anti-CD24 Percp-cy5.5, or anti-IL-10 PE (BioLegend, CA). To detect phosphorylation, single-cell suspensions of splenocytes were stimulated for 3 h with lipopolysaccharide (LPS; 10 μg/mL, Sigma, St. Louis) or for 30 min with rmIL-21 (100 ng/mL; R&D Systems). Cells were then fixed and permeabilized with BD Phosflow Lyse/Fix Buffer and Perm Buffer III (BD Biosciences), respectively. Finally, cells were stained with anti-B220 FITC, anti-CD5 BV421, anti-CD1d APC, or anti-pY727 PE. All data were analyzed using FlowJo software.
B cell stimulation
PBMCs isolated from patients or HCs were resuspended (at 2×106 cells/mL) in 48-well flat-bottom plates in culture medium and stimulated for 7 h with LPS (10 μg/mL; Sigma, St. Louis), phorbol 12-myristate 13-acetate (PMA, 20 ng/mL; Sigma, St. Louis), ionomycin (1 μg/mL; Sigma, St. Louis), and brefeldin A (BFA, 1×solution/mL; BioLegend, CA) before staining and flow cytometry analysis. Next, the cells were harvested and washed twice with PBS. Single-cell suspensions were then stained for 20 min on ice with predetermined optimal concentrations of anti-CD19 APC (BioLegend, CA). Stained cells were washed twice with PBS before fixation and permeabilization in Fixation and Permeabilization Buffer (BioLegend, CA). Finally, cells were stained for 30 min with anti-IL-10 PE (BioLegend, CA). To detect B10 cells in mice, single-cell suspensions of splenocytes were stimulated for 5 h with LPS, PMA (50 ng/mL), ionomycin (500 ng/mL), and BFA, followed by staining with anti-CD19 APC (BioLegend, CA) and anti-IL-10 PE (BioLegend, CA). For co-culture experiments, splenic B cells were purified using an EasySep™ Mouse B Cell Isolation Kit (STEMCELL Technologies, Canada). The cells (2×106 cells/mL) were then incubated for 48 h with mouse rmIL-21 (100 ng/mL, R&D Systems). After culture for 48 h, the IL-10 concentration in the supernatant was measured by ELISA (BioLegend, CA), and B10 cells was measured by flow cytometry analysis as described above.
Generation of bone marrow chimeras
Bone marrow was collected from DOCK8−/− (CD45.2+) mice and from C57BL/6 wild-type (CD45.1+) mice. For each chimera, CD45.2+ wild-type or DOCK8 KO bone marrow cells plus CD45.1+ bone marrow cells (4 × 106 cells in a 1:1 mixture) were transferred intravenously into lethally-irradiated (two doses of 550 rads each) wild-type CD45.1+ recipients. Recipient mice were allowed 8 weeks to reconstitute prior to challenge with ovalbumin (OVA).
Generation of OVA-induced allergic asthma model mice and nasal administration of recombinant IL-21
OVA-induced allergic asthma was elicited by sensitization with chicken OVA (5 μg, intraperitoneally (i.p.); Sigma-Aldrich) emulsified in 200 ml Imject Alum (Thermo Fisher Scientific) on Day 0, followed by two oropharyngeal aspiration challenges (on Days 14 and 21) with 1.5% OVA dissolved in 50 ml PBS. Control mice received only PBS. Mice were harvested 72 h after the second challenge[20]. Some mice received 20 ng of rmIL-21 (R&D Systems) into the nostrils (daily on Days 15–17, 18–20, or 15–20). This 3 day protocol was decided by conducting preliminary time-course experiments. The dose of rmIL-21 was obtained from a previous study[21]. As a negative control, groups of mice received neither sensitization nor challenge treatment, except for the final challenge on Day 21.
Adoptive cell transfer
Naïve splenic CD4+ T cells isolated from DOCK8 KO or wild-type mice were purified with the EasySep™ Mouse Naïve CD4+ T Cell Isolation Kit (STEMCELL Technologies, Canada). Next, 5 × 106 naïve CD4+ T cells were adoptively transferred into CD4KO mice via intravenous injection 1 day before immunization with OVA. OVA immunization was performed as described above. Recipient mice were analyzed on Day 21 post-OVA immunization.
Histopathological analysis
Lung tissue was harvested and fixed for 24 h in 10% formalin and then embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin. The degree of airway infiltration by inflammatory cells was scored by two independent investigators by double-blind screening. Peri-bronchiole and peri-vascular inflammation were evaluated using a scoring system of 0–4, where 0 represents no cells; 1, a few cells; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; and 4, a ring of inflammatory cells >4 cells deep.
Statistical analysis
GraphPad Prism 5 software was used for statistical analyses. Results are expressed as the mean ± SEM. The significance of differences between groups was determined using a two tailed unpaired Student’s t test. A P value <0.05 was deemed statistically significant.