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. 

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

OVA specific and total IgE production in serum and BALF of mouse asthma model