B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice were obtained by mating B-hBAFF/hBAFFR mice (111921) and B-hTWEAK/hAPRIL mice (110805). For validation data of this mouse model, you can refer to the validation data from the related gene humanized mouse models.

BAFF

• The exons 1-7 of mouse Baff gene that encode extracellular domain and part of transmembrane domain were replaced by human counterparts in B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice.
• The genomic region of mouse Baff gene that encodes cytoplasmic portion was retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The chimeric BAFF expression was driven by endogenous mouse Baff promoter, while mouse Baff gene transcription and translation will be disrupted.

BAFFR

• The exons 1-2 of mouse Baffr gene that encode extracellular domain and transmembrane domain were replaced by human counterparts in B-hCD3EDG/hBAFFR mice.
• The genomic region of mouse Baffr gene that encodes cytoplasmic portion was retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The chimeric BAFFR expression was driven by endogenous mouse Baffr promoter, while mouse Baffr gene transcription and translation will be disrupted.

TWEAK

• The genomic region of mouse Tweak gene that encodes the entire protein (from ATG to stop codon) were replaced with the corresponding human sequences 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.

APRIL

• The genomic region of mouse April gene that encodes the entire protein (from ATG to stop codon) were replaced with the corresponding human sequences in B-hTWEAK/hAPRIL mice.
• The promoter, 5’UTR region and 3’UTR region of the mouse gene were replaced by human counterparts. The human APRIL expression was driven by human APRIL promoter, while mouse April gene transcription and translation will be disrupted.

B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice (14431) were obtained by mating B-hBAFF/hBAFFR mice (111921) and B-hTWEAK/hAPRIL mice (110805).

• Mouse BAFF was detected in serum of wild-type C57BL/6JNifdc mice.
• Human BAFF was detected in serum of homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific BAFF expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice (female, 9-week-old, n = 3). Expression level of mouse and human BAFF were analyzed by ELISA (anti-Mouse BAFF ELISA kit: R&D Systems, MBLYS0; anti-Human BAFF ELISA kit: R&D Systems, DBLYS0B). Values are expressed as mean ± SEM.

• Mouse BAFFR was detectable on B cells in spleen of wild-type C57BL/6JNifdc mice.
• Human BAFFR was detectable on B cells in spleen of homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific BAFFR expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice. Protein expression was analyzed with anti-mouse BAFFR antibody (Biolegend, 134105) and anti-human BAFFR antibody (Biolegend, 316905) by flow cytometry.

• TWEAK was detectable in wild-type C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice due to the cross-reactivity of anti-TWEAK antibody.

Strain specific TWEAK expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice by western blot. Liver, spleen, lung and kidney were collected from wild-type C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice. Protein expression was analyzed with anti-TWEAK antibody (Proteintech, 12537-1-AP) by western blot.

• Human APRIL was detected in serum of homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific APRIL expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice (female, 9-week-old, n = 3). Expression level of human APRIL were analyzed by ELISA (anti-Human APRIL ELISA kit: thermofisher, BMS2008). Values are expressed as mean ± SEM.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice were similar to those in C57BL/6JNifdc mice.
• Humanization of BAFF, BAFFR, TWEAK, APRIL does not affect normal immune cell development or distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice (female, 9-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice were comparable to those in C57BL/6JNifdc mice.
• Humanization of BAFF, BAFFR, TWEAK, APRIL does not affect normal T cell development, differentiation, or distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from C57BL/6JNifdc mice and homozygous B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice (female, 9-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• No significant differences were observed compared with wild-type C57BL/6JNifdc mice.

Blood biochemical parameters of B-hBAFF/hBAFFR/hTWEAK/hAPRIL mice are shown. Values are expressed as mean ± SD.