Immunodeficiency 28 (IMD28) Cell Models for Research

Immunodeficiency 28 (IMD28) is a rare primary immunodeficiency disorder caused by mutations in the CD27 gene. The exact prevalence is unknown, but it is considered extremely rare, with fewer than 1 in 1,000,000 individuals affected. The condition is characterized by persistent symptomatic Epstein-Barr virus (EBV) infection, hypogammaglobulinemia, and an increased risk of lymphoma. Patients often present in childhood with recurrent infections, and the prognosis is variable, with some individuals developing life-threatening complications. Early diagnosis and management, including immunoglobulin replacement and hematopoietic stem cell transplantation, are critical for improving outcomes. The disease is inherited in an autosomal recessive pattern, and genetic testing is essential for confirmation.

IMD28 provides a unique opportunity to study the role of CD27 in immune regulation and EBV control. CD27 is a costimulatory molecule expressed on T cells and natural killer (NK) cells, and its interaction with CD70 is crucial for T cell activation and B cell differentiation. The disease serves as a natural model for understanding the molecular mechanisms underlying EBV susceptibility and lymphomagenesis. Research using patient-derived cells and gene-edited models can elucidate the signaling pathways downstream of CD27, identify potential therapeutic targets, and contribute to the development of targeted therapies. The rarity of the disease underscores the importance of robust in vitro models for mechanistic studies and drug screening.

IMD28 is primarily an immunodeficiency, but the increased risk of lymphoma highlights the involvement of oncogenic pathways. The major pathways implicated include:

• • NF-κB signaling: CD27 activates NF-κB via TRAF2 and TRAF5, leading to the expression of anti-apoptotic genes. Loss of CD27 impairs this pathway, leading to defective T cell survival and proliferation.
• • T cell receptor (TCR) signaling: CD27 costimulation enhances TCR-mediated signaling, promoting T cell activation and effector function. In its absence, T cell responses are suboptimal, leading to impaired immune surveillance.
• • EBV-driven B cell transformation: EBV infection of B cells in the absence of effective T cell control can lead to uncontrolled B cell proliferation and lymphomagenesis. The molecular mechanisms involve viral oncoproteins such as LMP1 and EBNA2, which activate NF-κB and JAK/STAT pathways.

Data from ClinVar and the Human Gene Mutation Database (HGMD) indicate that over 30 distinct pathogenic mutations in CD27 have been reported. These mutations are typically biallelic and result in complete loss of CD27 function.

The loss of CD27 disrupts several signaling networks:

• • NF-κB pathway: Reduced activation of canonical NF-κB, leading to decreased expression of anti-apoptotic genes (e.g., BCL2, BCL-XL) and impaired T cell survival.
• • PI3K/AKT pathway: CD27 costimulation activates PI3K, promoting AKT phosphorylation and cell survival. Loss of CD27 reduces AKT activity, affecting T cell metabolism and function.
• • JAK/STAT pathway: CD27 signaling can modulate cytokine responses, and its absence may alter STAT activation, impacting T cell differentiation.
• • Co-stimulatory molecules: CD27 is part of the TNF receptor superfamily; its loss may affect the expression of other co-stimulatory molecules, leading to broader immune dysregulation.

Organoid models, such as tonsil organoids, can be used to study EBV infection and immune responses in a more physiologically relevant context. However, gene-edited organoids are still in development for IMD28.

• • CD27 knockout mice: These mice exhibit impaired T cell responses and increased susceptibility to viral infections, but they do not fully recapitulate the human phenotype.
• • EBV-infected humanized mice: These mice are engrafted with human immune cells and can be infected with EBV, providing a model to study the role of CD27 in EBV control.
• • Patient-derived xenografts (PDX): Rarely used due to the rarity of the disease, but can be generated from patient lymphomas.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with precise CD27 mutations. For example, a CD27 knockout cell line can be generated in a T cell line like Jurkat, providing a model to study the loss of CD27 function. Alternatively, a knock-in cell line with a specific pathogenic mutation (e.g., p.Cys53Tyr) can be created to study the impact of that mutation. These models are commercially available from various sources, and they are sequence-verified to ensure the desired edit. Such gene-edited cell models are invaluable for functional studies, drug screening, and understanding the molecular mechanisms of IMD28.

Gene-edited cell lines with CD27 knockout or knock-in mutations are used to validate the function of CD27 in immune signaling. For example, researchers can compare the expression of downstream targets (e.g., BCL2, IL-2) between wild-type and CD27 knockout cells to identify CD27-dependent genes. Additionally, these models can be used to study the interaction between CD27 and its ligand CD70, and to screen for small molecules that can rescue CD27 function.

Isogenic pairs (wild-type vs. CD27 knockout) are used in high-throughput screens to identify compounds that selectively kill CD27-deficient cells, which may have therapeutic potential for IMD28-associated lymphomas. Furthermore, these models can be used to study resistance mechanisms to immunomodulatory drugs, as CD27 signaling may influence drug sensitivity.

CRISPR-based synthetic lethality screens using CD27 knockout cells can identify genes that are essential for the survival of CD27-deficient cells. These genes could serve as novel therapeutic targets or biomarkers for disease progression. Additionally, transcriptomic and proteomic profiling of gene-edited cells can reveal biomarkers associated with CD27 loss.