CD40L deficiency is a well-described inborn error of immunity (IEI). The disease is characterized immunologically by impaired class-switching and T cell costimulation. A male infant presented at 8 months of age with recurrent cellulitis. At 11 months, he was admitted for prolonged fever and leukopenia with vomiting and watery diarrhea, periorbital edema, back abscess, increased sIL2R at 50,598 pg/mL (<14,491), and normal CXCL9 at 22 pg/mL with mild to modest increases in IL-18 at 694 pg/mL (<185.4), IL-6 at 226.4 pg/mL (<10), serum amyloid A (SAA) at 1,413 mcg/mL (<373.8), IL-8 at 196 (<10), and IL-10 at 101pg/mL (<10). His IgG and IgA were low at <30 and <10 mg/dL, respectively. He developed Pseudomonas sepsis, palatal ulceration, jejunal perforation requiring repair, Candida peritonitis, persistent neutropenia, CMV, and Parechovirus viremia. Along with the hypogammaglobulinemia, class-switched memory B cells were decreased for age. Flow cytometry was performed for CD40L expression on activated CD4+ T cells along with functional evaluation of ligand binding to a soluble form of the receptor (CD40), using CD40-muIg. There was significantly diminished CD40L expression with essentially absent binding to the receptor. Genetic testing confirmed a diagnosis of CD40L deficiency due to a hemizygous variant in CD40LG, c.674T>C; p.Leu225Ser (NM_000074.2). We had previously evaluated a 4-year-old male with intermittent, non-bloody diarrhea, pneumonia, hypogammaglobulinemia, and significant neutropenia, and identified this novel variant, now also observed in this infant. We utilized advanced computational tools to evaluate the molecular impact of this variant on the structure and function of CD40L. The Leu225Ser variant is located within the trimerization interface of CD40L. Molecular modeling, mutagenesis alanine scanning, and molecular dynamic simulations revealed that the variant causes loss of critical intramolecular time-dependent interactions between hydrophobic molecules that would typically facilitate trimerization of wild-type CD40L for optimal interaction with the CD40 receptor. This analysis of the Leu225Ser variant revealed the molecular basis of the decreased CD40L protein expression (loss of contact to form trimers) and impaired CD40 binding (loss of intramolecular bonds affecting folding and impaired interaction at the trimerization interface). The patient also had immune paralysis due to decreased MHC class II (HLA-DR) expression on CD14+ monocytes, a result of a compensatory anti-inflammatory response syndrome (CARS). Assessment of neutrophil activation and granule markers (CD66b, lactoferrin, myeloperoxidase [MPO], and elastase) revealed low-normal CD66b and lactoferrin with slightly increased MPO and elastase, suggesting a complex and dysfunctional neutrophil response rather than simple activation or deactivation. This phenotype is consistent with the known pathology of CD40L deficiency, in which neutrophils are defective in their ability to respond effectively to infection, leading to chronic inflammation and tissue damage despite persistent signs of granule release. Treatment with rIFNg has been shown to reverse neutrophil dysfunction in X-linked hyper-IgM syndrome (XHIGM) but also restore HLA-DR expression on monocytes and reverse immune paralysis. He is currently being evaluated for hematopoietic cell transplantation (HCT). This case exemplifies the relevance of utilizing a variety of strategies to evaluate novel variants in IEI genes. Further, molecular and computational studies permitted an improved understanding of the impact of the Leu225Ser variant on CD40L protein expression and function.
