The focus of the laboratory is threefold: 1) delineate the genetic basis of immune deficiency, 1) study the consequence of these gene mutations on the ontogeny of hematopoetic cells, and 3) explore therapies for these disorders based on an understanding of the underlying molecular mechanism of disease. Employing the candidate genetic approach, we identified mutations in the gene encoding NEMO (NF- kB essential modulator), an intracellular signaling constituent of the NF-kB pathway, results in ectodermal dysplasia with an immune defect in B cell terminal differentiation. Mutations in the zinc finger domain of NEMO block CD40 mediated activation of NF- kB and prevent B cells from undergoing class switch recombination (CSR) and APC?s from synthesizing NF -kB regulated cytokines such as IL -12 or TNF-a when stimulated with CD40 ligand. Interestingly, NF- KB activation via other signaling pathways such as TNF-a or members of the Toll- like receptor family are intact in these patients. We reason that the inherent immune deficiency in patients with hypomorphic mutations in NEMO is directly attributable to defective signaling via NF-kB, and would be accompanied by differences in target gene expression. We have previously compared gene expression profiles of B cells from patients with CD40 ligand deficiency and ectodermal dysplasia with immune deficiency (EDA- ID) as a means to identify NF -kB target genes mediated by CD40L signaling, which modulate CSR. We show XHM -ED patients are broadly defective in gene expression programs controlling proliferation, cell cycle progression, repair and ligation of DNA ends, and mRNA processing. We now have expanded our studies on gene expression profiling to dendritic cells. In tissue culture experiments, DCs are typically several orders of magnitude more effective as antigen presenting cells than B cells or macrophages. In addition, during contact allergy and transplantation DCs migrate from peripheral tissues to lymphoid organs, the sites for the induction of most types of primary immune responses. Preliminary data from our lab indicates that missense mutations in Exon 10 of NEMO are permissive for triggered activation of Rel A, but are defective in the activation of the NF-kB subunit c-Rel. In contrast to normal controls, the DCs from EDA-ID patients after stimulation with CD40L are unable to induce proliferation or cytokine production from alloreactive T cells. With gene expression profiling, we expect to produce a reasonably complete picture of gene regulation mediated by the transcription factor c-Rel in DCs and its influence on primary immune responses. Our approach of utilizing comparative genomics technologies to the study of this rare genetic disease, has allowed us to deconstruct a complex signaling pathway essential for B cell terminal differentiation, and dendtric cell activation. The long-range goal is to dissect the physiologic role of CD40 intracellular signaling and its influence in humoral and cell mediated immunity. The protein CYLD is a member of the de-ubiquitinating enzyme family of proteins that has been shown to deubiquitinate NEMO by direct interaction and inhibit of NF-kB activation. The laboratory is working on the CYLD knockout mouse. The primary aim of these experiments is to further elucidate the developmental and functional role of NEMO associated proteins in lymphocytes and APC?s.
