mRNA expression analysis
Strain specific analysis of IL4 and IL4RA gene expression in WT and B-hIL4/hIL4RA mice by RT-PCR. Mouse Il4 and Il4ra mRNA was detectable in splenocytes of wild-type (+/+) but not in homozygous B-hIL4/hIL4R mice (H/H) mice. Human IL4 and IL4RA mRNA was detectable only in homozygous B-hIL4/hIL4R mice but not in wild-type mice.
Strain specific IL4 expression analysis in homozygous B-hIL4/hIL4RA mice by ELISA.
Serum were collected from WT and homozygous B-hIL4/hIL4RA (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by ELISA with species-specific IL4 ELISA kit. Mouse Il4 was detectable in WT mice. Human IL4 was exclusively detectable in homozygous B-hIL4/hIL4RA mice but not WT mice.
Analysis of spleen leukocyte subpopulations by FACS
Analysis of subpopulation of T cells in lymph node by FACS
(A) Splenic B cells from C57BL/6 and B-hIL4/hIL4RA mice were cultured with LPS alone or together with 50ng/mL mIL-4/hIL-4. Culture supplements were harvested on day 6 for quantification of IgE by ELISA.(B) Splenic B cells from B-hIL4/hIL4RA mice were incubated with increasing doses of dupilumab (in house) for half of an hour before adding LPS and hIL-4 (50 ng/mL). Culture supernatant was harvested on day 6 for quantification of IgE by ELISA. The result show that B cells from B-hIL4/hIL4RA mice responded well to LPS and IL-4 with IgE production, as similar to C57BL/6 mice. Dupilumab (in house) could effectively block the expression of IgE.
IL-4 induced IgE secretion in B-hIL4/hIL4RA mice
Analysis of activity of B cells in spleen by FACS
Splenocytes were isolated from B-hIL4/hIL4RA mice and incubated with varying concentrations of IgG4 or dupilumab for half an hour. Adding human IL4 (50 pM) for 72 hours and analyzing the activity of B cells. The dupilumab group showed an effect to block B cell activation in B-hIL4/IL4RA mice and showed a dose-dependent effect; the control hIgG4 did not show an effect on block B cell activation.
The number of BALF immune cells in mouse asthma model