OX40L: Enhances effector T cell function, sustains their survival and suppresses apoptosis 

• Gene Information: Tumor Necrosis Factor Ligand Superfamily Member 4 (TNFSF4, also known as OX40L) is a protein-coding gene located on chromosome 1q25.1. OX40L is a 35 kD member of the TNF superfamily and is also known as OX40 ligand, CD252, and CD134L. 
• Protein Expression: OX40L functions as a type II transmembrane glycoprotein. Beyond abundant expression in activated B cells and antigen presenting cells, OX40L can be detected in the heart, bone and lung, while activated T cells express this molecule at a low level. 
• Signaling Pathway: The OX40L–OX40 signaling axis drives persistent activation and proliferation of effector T cells and effector memory T cells, while remaining quiescent in naive T cells and resting memory T cells. Engagement of OX40 with its ligand OX40L promotes the differentiation of both Th1 and Th2 effector cells. 
• Therapeutic Inhibition: Recent therapeutic success in clinical trials highlights the importance of the OX40L–OX40 axis as a promising target for the treatment of atopic dermatitis.

OX40 

• The exons 1-5 of mouse OX40 gene that encode the extracellular domain were replaced by human OX40 exons 1-5 in B-hOX40/hOX40L mice(C). 
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human OX40 expression to be driven by the native mouse Ox40 promoter, while endogenous mouse Ox40 transcription and translation are abolished. 

OX40L 

• The exons 2-3 of mouse Ox40l gene that encode the extracellular region were replaced by human OX40L exons 2-3 in B-hOX40/hOX40L mice(C). 
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human OX40L expression to be driven by the native mouse Ox40l promoter, while endogenous mouse Ox40l transcription and translation are abolished. 
• B-hOX40/hOX40L mice(C) were obtained by breeding B-hOX40 mice(C) with B-hOX40L mice(C).

Strain specific OX40 expression analysis in homozygous B-hOX40/hOX40L mice(C) by flow cytometry. Splenocytes were collected from wild-type BALB/cCrSlcNifdc mice(+/+) and homozygous B-hOX40/hOX40L mice(C) (H/H;H/H) stimulated with anti-CD3ε in vivo, and protein expression was analyzed with anti-mouse OX40 antibody (Biolegend, 119414)  and anti-human OX40 antibody (Biolegend, 350004) by flow cytometry. mOX40 was only detectable in T cells of wild-type BALB/cCrSlcNifdc mice. hOX40 was only detectable in T cells of homozygous B-hOX40/hOX40L mice(C), but not in wild-type BALB/cCrSlcNifdc mice.

Strain specific OX40L expression analysis in homozygous B-hOX40/hOX40L mice(C) by flow cytometry. Bone marrow cells were collected from wild-type BALB/cCrSlcNifdc mice(+/+) and homozygous B-hOX40/hOX40L mice(C)(H/H;H/H). BMDCs were induced from bone marrow cells and stimulated with LPS in vitro. Then BMDCs were analyzed with anti-mouse OX40L antibody (Biolegend, 108814)  and anti-human OX40L antibody (BD Horizon™, 563766) by flow cytometry. mOX40L was only detectable in BMDCs of wild-type BALB/cCrSlcNifdc mice. hOX40L was only detectable in BMDCs of homozygous B-hOX40/hOX40L mice, but not in wild-type BALB/cCrSlcNifdc mice.

Strain specific cytokines expression analysis in wild-type BALB/cCrSlcNifdc mice and homozygous humanized B-hOX40/0X40L mice(C) by ELISA. Serum was collected from wild-type BALB/cCrSlcNifdc mice (+/+) (female, n=2, 7-week-old) and homozygous B-hOX40/0X40L mice(C) (H/H;H/H)  (female, n=3, 7-week-old) stimulated with anti-mouse CD3ε antibody (37.5 μg/mL, 200 μL/mouse, i.p.) for 2 hrs in vivo. Expression level of IL4, IL13, IL2, IL22, IL17A, and IFN-γ were analyzed by ELISA. After mCD3ε stimulation, a significant increase of mouse IL4, IL13, IL2, IL22, IL17A, and IFN-γ were detected in BALB/cCrSlcNifdc mice (n=3) and homozygous B-hOX40/0X40L mice(C)  (n=3). Values are expressed as mean ± SEM.

Experimental schedule for induction of AD model and in vivo efficacy of anti-human OX40L antibody. OXA was applied to dorsal and ear skin of mice on day 0, and then challenge to the same site of skin nine times from days 7 to 18. Amlitelimab analog (in house) was administered by intraperitoneal injection twice a week on days -1 to 16. Serum was collected at the endpoint on day 19. AD: atopic dermatitis; OXA: oxazolone.

Efficacy of anti-human OX40L antibody in B-hOX40/hOX40L mice(C). Mice in each group were intraperitoneally injected with anti-hOX40L antibody (in house, n=7). (A) Body weight changes during the treatment. (B) Statistical analysis of ear thickness in each group. (C) Total IgE levels in serum. The results showed that compared to the untreated group (G2), the group of mice treated with anti-OX40L antibody showed a significant reduction in ear thickness. Serum was collected at the study endpoint. IgE level was analyzed by ELISA. The results showed that the levels of total IgE in mice treated with anti-OX40L antibody was lower than that in untreated mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.