Gene targeting strategy for B-hTWEAK/hAPRIL mice.

The genomic region of mouse Tweak gene that encodes the full-length protein was replaced by human counterparts in B-hTWEAK/hAPRIL mice. The promoter and 5’UTR region of the mouse gene were retained. The 3’UTR region of the mouse gene was replaced by human counterparts. The human TWEAK expression was driven by endogenous mouse Tweak promoter, while mouse Tweak gene transcription and translation will be disrupted.

The genomic region of mouse April gene that encodes the full-length protein was replaced by human counterparts in B-hTWEAK/hAPRIL mice. The promoter, 5’UTR region and 3’UTR region of the mouse gene was replaced by human counterparts. The human APRIL expression was driven by endogenous mouse April promoter, while mouse April gene transcription and translation will be disrupted.

Strain specific analysis of TWEAK and APRIL gene expression in wild type (WT) mice and B-hTWEAK/hAPRIL mice by RT-PCR. Mouse Tweak and April mRNA were detectable only in lung of WT mice (+/+). Human TWEAK and APRIL mRNA was detectable only in homozygous B-hTWEAK/hAPRIL mice (H/H) but not in WT mice (+/+).

Strain-specific APRIL expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTWEAK/hAPRIL mice by ELISA. Serum were collected from wild-type C57BL/6JNifdc mice (+/+) (n=3, 7 weeks old, male) and homozygous B-hTWEAK/hAPRIL mice (H/H) (n=3, 7 weeks old, male) stimulated with LPS in vivo (200 μg/mouse, stimulation for 4 hours, i.p.). Expression level of human APRIL were analyzed by ELISA (human APRIL ELISA kit: thermofisher, BMS2008). Human APRIL was exclusively detectable in homozygous B-hTWEAK/hAPRIL mice, but not in wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM.

Strain specific TWEAK expression analysis in homozygous B-hTWEAK/APRIL mice by western blot. Lung was collected from wild type (WT) mice (+/+) and homozygous B-hTWEAK/APRIL mice (H/H), and analyzed by western blot with anti-TWEAK antibody. TWEAK was detectable in WT mice (+/+) and homozygous B-hTWEAK/APRIL mice (H/H) due to the cross-reactivity of antibodies.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice (female, 8-week-old, n=3) and homozygous B- hTWEAK/hAPRIL 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, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hTWEAK/hAPRIL mice were similar to those in C57BL/6JNifdc mice, demonstrating that humanization of TWEAK and APRIL does not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6JNifdc mice (female, 8-week-old, n=3) and homozygous B-hTWEAK/hAPRIL mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hTWEAK/hAPRIL mice were similar to those in C57BL/6JNifdc mice, demonstrating that humanization of TWEAK and APRIL does not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in lymph nodes by flow cytometry. Leukocytes were isolated from wild-type C57BL/6JNifdc mice (female, 8-week-old, n=3) and homozygous B-hTWEAK/hAPRIL mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hTWEAK/hAPRIL mice were similar to those in C57BL/6JNifdc mice, demonstrating that humanization of TWEAK and APRIL does not change the frequency or distribution of these cell types in lymph nodes.. Values are expressed as mean ± SEM.