CD40/CD40L: A key costimulatory axis in adaptive immunity and its therapeutic intervention

• Gene Information: CD40 is a protein-coding gene located on chromosome 20q13.12, encoding a cell surface receptor that belongs to the tumor necrosis factor receptor (TNFR) superfamily. Its corresponding ligand, CD40L (CD154), is encoded by a gene situated on chromosome Xq26.3 and is a member of the TNF superfamily.

• Protein Expression: CD40 is constitutively expressed primarily by antigen-presenting cells (APCs) including B cells, dendritic cells, and macrophages, as well as endothelial cells. CD40L is predominantly and transiently expressed on the surface of activated T helper cells and platelets during an immune response.

• Signaling Pathway: CD40L exerts its effects by binding as a functional homotrimer to CD40 receptors on target cells, inducing receptor clustering. This interaction recruits intracellular tumor necrosis factor receptor-associated factors (TRAFs), activating downstream canonical/non-canonical NF-κB and MAPK pathways to drive B cell activation, antibody class switching, and macrophage differentiation.

• Therapeutic Intervention: By selectively modulating this costimulatory axis, antagonistic therapeutics (such as dapirolizumab or frexalimab) block CD40/CD40L binding to inhibit pathogenic autoantibody production and systemic inflammation in autoimmune diseases like lupus or multiple sclerosis. Alternatively, agonistic anti-CD40 antibodies (such as selicrelumab) are utilized in immuno-oncology to "license" dendritic cells and unleash cell-mediated anti-tumor immunity.

CD40

• The exons 2-7 of mouse Cd40 gene that encodes the extracellular region were replaced by human CD40 exons 2-7 in B-hCD40/hCD40L/hFcRn mice.

• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human CD40 expression to be driven by the native mouse Cd40 promoter, while endogenous mouse Cd40 transcription and translation are abolished.

CD40L

• The exons 2-5 of mouse Cd40l gene that encode the extracellular region were replaced by human CD40L exons 2-5 in B-hCD40/hCD40L mice.

• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human CD40L expression to be driven by the native mouse Cd40l promoter, while endogenous mouse Cd40l transcription and translation are abolished.

FcRn

• The human full length FCGRT cDNA sequence was inserted in the Fcgrt exons 2-4 in B-hCD40/hCD40L/hFcRn mice.

B-hCD40/hCD40L/hFcRn Mice were obtained by mating B-hCD40/hCD40L Mice (121335) and B-hFcRn Mice (110001).

Strain specific analysis of CD40 expression in homozygous B-hCD40/hCD40L/hFcRn mice by flow cytometry. Splenocytes were collected from C57BL/6 mice(+/+;+/+) and homozygous B-hCD40/hCD40L/hFcRn mice (H/H;H/H) stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hr, and analyzed by flow cytometry with species-specific anti-CD40 antibody. Mouse CD40 was detectable in wild-type mice. Human CD40 was exclusively detectable in homozygous B-hCD40/hCD40L/hFcRn mice but not in wild-type mice.

Strain specific CD40L expression analysis in wild-type C57BL/6 mice and homozygous B-hCD40/hCD40L/hFcRn mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice(+/+) and homozygous B-hCD40/hCD40L/hFcRn mice (H/H;H/H;H/H) and stimulated with PMA & Ionomycin. Protein expression was analyzed with anti-mouse CD40L antibody (Biolegend, 106509) and anti-human CD40L antibody (Biolegend, 310805) by flow cytometry. Mouse CD40L was detectable in wild-type C57BL/6 mice. Human CD40L was exclusively detectable in homozygous B-hCD40/hCD40L/hFcRn mice but not in wild-type C57BL/6 mice.

Strain specific analysis of FcRn expression in homozygous B-hCD40/hCD40L/hFcRn mice by western blot. Heart, liver, spleen, lung, kidney, and colon were collected from C57BL/6 mice (+/+;+/+) and homozygous B-hCD40/hCD40L/hFcRn mice (H/H;H/H;H/H) and analyzed by western blot with species-specific anti-FcRn antibody. Mouse FcRn was detectable in wild-type mice. Human FcRn was detectable only in homozygous B-hCD40/hCD40L/hFcRn mice but not in wild-type mice.