Strain specific analysis of KITLG mRNA expression in wild-type C57BL/6 mice and homozygous B-hKIT/hKITLG mice by RT-PCR. Lung RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hKIT/hKITLG mice (H/H;H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human KITLG primers. Mouse Kitlg mRNA was only detectable in wild-type mice but not in homozygous B-hKIT/hKITLG mice. Human KITLG mRNA was only detectable in homozygous B-hKIT/hKITLG mice but not in wild-type mice.

Strain specific KITLG expression analysis in homozygous B-hKITLG mice by ELISA. Serum was collected from wild-type mice (+/+) and homozygous B-hKITLG mice (H/H), and analyzed by ELISA with species-specific KITLG ELISA kit. Mouse KITLG was detectable in wild-type mice. Human KITLG was exclusively detectable in homozygous B-hKITLG mice but not in wild-type mice.

Strain specific KIT expression analysis in homozygous B-hKIT/hKITLG mice by flow cytometry. Bone marrow were collected from wild-type mice (+/+) and homozygous B-hKIT/hKITLG mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-KIT antibody. Mouse KIT was detectable in wild-type mice. Human KIT was exclusively detectable in homozygous B-hKIT/hKITLG mice but not in wild-type mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hKIT/hKITLG mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hKIT/hKITLG  mice were similar to those in C57BL/6 mice, demonstrating that humanization of KIT and KITLG does not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in blood and lymph node of B-hKIT/hKITLG mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM.

The KIT/KITLG interaction plays a critical role in mast cell homeostasis. Flow cytometry was used to analyze differences in mast cell survival between wild-type C57BL/6JNifdc mice and heterozygous B-hKIT/hKITLG mice. Peritoneal lavage was collected from wild-type C57BL/6JNifdc (+/+) mice and heterozygous B-hKIT/hKITLG mice (H/+; H/+) and analyzed by flow cytometry using anti-mouse KIT antibody (Biolegend, 105835) and anti-human KIT antibody (Biolegend, 313203). Compared with mouse mast cells (FcɛRIα+ KIT+) in C57BL/6JNifdc mice, mast cells were normally present in the peritoneal lavage of  heterozygous B-hKIT/hKITLG mice.