TWEAK: A TNF ligand regulating tissue remodeling and inflammation.

APRIL: A cytokine driving B-cell survival and humoral immunity.

• Gene Information: TNFSF12 (TWEAK) and TNFSF13 (APRIL) are located on chromosome 17p13, encoding structurally distinct cytokine ligands belonging to the tumor necrosis factor (TNF) superfamily.
• Protein Expression: TWEAK is widely expressed in endothelial cells, monocytes, and injured tissues, while APRIL is predominantly produced by immune cells like myeloid cells, T cells, and dendritic cells.
• Signaling Pathway: TWEAK signals exclusively through the Fn14 receptor to trigger NF-κB pathways, whereas APRIL binds BCMA and TACI receptors to stimulate critical B-cell survival and differentiation cascades.
• Therapeutic Inhibition: Targeting the TWEAK/Fn14 axis helps mitigate chronic fibroinflammatory autoimmune diseases, while therapeutic inhibition of APRIL effectively suppresses pathogenic antibody production in lupus nephritis and IgA nephropathy.

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.

• Mouse Tweak and April mRNA were detected in lung of wild-type C57BL/6 mice.
• Human TWEAK and APRIL mRNA were detected in homozygous B-hTWEAK/hAPRIL mice, but not in wild-type C57BL/6 mice.

Species specific analysis of TWEAK and APRIL gene expression in wild-type C57BL/6 mice and homozygous humanized B-hTWEAK/hAPRIL mice by RT-PCR. Lung RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTWEAK/hAPRIL mice (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Tweak primers, human TWEAK primers, mouse April primers, and human APRIL primers.

• Human APRIL was detected in serum of homozygous B-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-hTWEAK/hAPRIL mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice and homozygous B-hTWEAK/hAPRIL mice (male, 6-week-old, n = 3) stimulated with 200 μg/mouse LPS for 4 hours. Expression level of human APRIL were analyzed by ELISA (anti-human APRIL kit: thermo fisher, BMS2008). Values are expressed as mean ± SEM. ND: not detectable.

• TWEAK was detectable in lung of homozygous B-hTWEAK/hAPRIL mice and wild-type C57BL/6 mice due to the cross-reactivity of antibodies.

Strain specific TWEAK expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hTWEAK/hAPRIL mice by western blot. Lung were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hTWEAK/APRIL mice (H/H). Protein expression was analyzed with anti-TWEAK antibody (thermo fisher, PA5-96379) by western blot.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hTWEAK/hAPRIL mice were similar to those in C57BL/6JNifdc mice.
• Humanization of TWEAK and 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-hTWEAK/hAPRIL mice (female, 8-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-hTWEAK/hAPRIL mice were comparable to those in C57BL/6JNifdc mice.
• Humanization of TWEAK and 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-hTWEAK/hAPRIL mice (female, 8-week-old, n = 3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Keyhole limpet hemocyanin (KLH) induced T-cell dependent antibody response (TDAR) in B-hTWEAK/hAPRIL mice. B-hTWEAK/hAPRIL mice were immunized by intraperitoneal injection of KLH/CFA suspension on day 1. Blood samples were collected on days 14 and 21, and KLH-specific IgG and IgM levels were detected by ELISA.

Evaluation of KLH-induced TDAR in B-hTWEAK/hAPRIL mice. (A) Body weight changes during treatment. (B) Mouse KLH-IgM and KLH-IgG on Day 14. (C) Mouse KLH-IgM and KLH-IgG on Day 21. The levels of KLH-specific IgG and IgM in B-hTWEAK/hAPRIL mice increased after KLH immunization, indicating that T-cell dependent antibody response (TDAR) was successfully established using B-hTWEAK/hAPRIL mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.