Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a p50 DNA binding protein and a p65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. However, this transcription factor plays a role in the induction of diverse sets of genes throughout the body in response to hundreds of different inducers. While studying a rare clinical condition of immunodeficiency, we have discovered the first germline mutation in CARD11, a protein that forms a vital signaling link between the antigen receptor in both B and T lymphocytes and the induction of NF-kappaB. We uncovered CARD11 mutations in one family with congenital lymphoid hyperplasia first reported in The New England Journal of Medicine in 1971 as well as in a child adopted from China in a second family. The affected family members exhibit excessive accumulation of poorly differentiated B lymphocytes but not T lymphocytes. The dominant missense mutations that we identified cause constitutively activated NF-kappaB contributing to downstream proliferation in B cells. However, the mutant CARD11 protein causes non-responsiveness or anergy in T cells resulting in poor IL-2 production and proliferation. Thus, we have identified the underlying genetic cause of this hereditary B cell disorder and have potentially uncovered a new molecular explanation for why CARD11 mutations predispose to B but not T lymphoid malignancies. This can be understood in terms of the 2-signal model for T cell activation in which T lymphocytes require stimulation through both the antigen receptor (signal 1) and a costimulatory receptor (signal 2) in order to induce proliferation. A corollary is that the provision solely of signal 1 in T cells leads to poor responsiveness or anergy. We observed this phenomenon in our patients, since E127G CARD11 causes internal constitutive activation of NF-kappaB, an important feature of signal 1, in the absence of a concomitant signal 2. This likely accounts for the accumulation of poorly responsive, anergic T cells. By contrast, B cell proliferation can be triggered by BCR crosslinking alone, which is mimicked by mutant CARD11-driven NF-kappaB activity. We posit that a chronic TCR-like signal 1 provided through mutant CARD11 can be converted to a proliferative signal for the patients T cells in vivo when proper costimulation (signal 2) is provided by professional antigen presenting cells. Defects in T cell help to B cells, related to T cell hyporesponsiveness, may partly explain the paucity of germinal centers and autoimmune manifestations we observed in these patients. On the other hand, deficiencies in T cell-independent humoral responses to polysaccharide antigens also point to intrinsic defects in B cell signaling and effector function with E127G CARD11 present. Our discovery of a germline gain-of-function mutation in CARD11 illuminates how antigen receptor signaling is regulated differently in B and T cells, even though the proximal signaling machinery is very similar. This surprising difference has not been revealed in somatic CARD11 mutations in diffuse large cell B cell lymphoma, in which only B cells harbor the mutation but it can potentially explain the preponderance of B cell rather than T-cell lymphomas associated with frequently-occurring activating somatic mutations in this gene. Our molecular analysis of this autosomal dominant lymphoproliferative disorder, which we call B cell expansion with NF-kappaB and T cell anergy or BENTA disease may represent a novel precursor state for B cell malignancies like B-chronic lymphocytic leukemia. The disease also may reveal how dysregulation of NF-kapppaB via CARD11 may predispose to selective proliferation and differentiation arrest in B cells, but defective proliferation and function of T cells. By re-examining the molecular basis of a rare genetic disorder first reported for decades ago, our discovery may open new avenues to treat this lymphoproliferative disease and potentially prevent the development of lymphoma by using agents that can block the overactive NF-kappaB. We continue to investigate the molecular abnormalities in BENTA patients. To gain insight into the biochemical regulation of NF-kapppaB, we have purified to homogeneity the human versions of the Rel homology domains (RHD) of subunits of the NF-kapppaB complex. We then studied the subunit interactions between the p50 and p65 RHD species and found that there was a dramatic preference of the p50/p65 RHD heterodimers compared to either the p50 or p65 RHD homodimers. From these studies, we deduce that the relative levels of Rel proteins present in a cell may be a means for modulating the populations of various Rel dimer species, in addition to well-characterized mechanisms like phosphorylation or cellular localization (e.g. export from or import into the nucleus). Studies of the DNA-binding properties of these RHD complexes revealed their sequence-specificity, and yielded insights into the mechanism by which the p50 and p65 NF-B subunits recognize specific DNA elements. These results promise to provide insights into mammalian gene expression and, given the importance of NF-kapppaB for the proper induction and transcription for genes involved in immunity, may shed light on specific gene regulation in immune system diseases. We have also investigated the role of microRNAs (miRNAs) in gene regulatory processes. In particular, we have studied the effects of the deficiency of Dicer which is essential for embryonic development and embryonic stem cell (ESC) proliferation and pluripotency. We found that Dicer and ESC-specific miRNAs, belonging to the miR-290 family, are important in silencing differentiation genes, such as the Hox genes, by regulating a pathway that coordinates proper Polycomb localization. Introduction of mature miR-291 into Dicer-deficient ESCs restores Polycomb-mediated silencing of Hox genes. This effect is mediated through a molecular cascade involving the methyltransferases Ash1l and Whsc1, whose expression is regulated by miR-290. Knockdown of Ash1l and Whsc1 partially prevented aberrant Hox gene expression in Dicer-deficient ESCs. Collectively, our data revealed that targets of ESC-specific miRNAs control Polycomb accumulation and thereby function to inhibit precocious ESC differentiation. We hope that further investigations may shed light on whether miRNAs play a role in genetic diseases involving NF-kapppaB as well as other aspects of the immune signaling system.
| Comrie, William A; Faruqi, Aiman J; Price, Susan et al. (2018) RELA haploinsufficiency in CD4 lymphoproliferative disease with autoimmune cytopenias. J Allergy Clin Immunol 141:1507-1510.e8 |
| Grönholm, Juha; Pagni, Philippe P; Pham, Minh N et al. (2017) Metabolically inactive insulin analogue does not prevent autoimmune diabetes in NOD mice. Diabetologia 60:1475-1482 |
| Ozen, Ahmet; Comrie, William A; Ardy, Rico C et al. (2017) CD55 Deficiency, Early-Onset Protein-Losing Enteropathy, and Thrombosis. N Engl J Med 377:52-61 |
| Lenardo, Michael; Lo, Bernice; Lucas, Carrie L (2016) Genomics of Immune Diseases and New Therapies. Annu Rev Immunol 34:121-49 |
| Lucas, Carrie L; Lenardo, Michael J (2015) Identifying genetic determinants of autoimmunity and immune dysregulation. Curr Opin Immunol 37:28-33 |
| Kanellopoulou, Chryssa; Gilpatrick, Timothy; Kilaru, Gokhul et al. (2015) Reprogramming of Polycomb-Mediated Gene Silencing in Embryonic Stem Cells by the miR-290 Family and the Methyltransferase Ash1l. Stem Cell Reports 5:971-978 |
| Chaigne-Delalande, Benjamin; Li, Feng-Yen; O'Connor, Geraldine M et al. (2013) Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D. Science 341:186-91 |
| Lo, Bernice; Ramaswamy, Madhu; Davis, Joie et al. (2013) A rapid ex vivo clinical diagnostic assay for fas receptor-induced T lymphocyte apoptosis. J Clin Immunol 33:479-88 |
| Pandiyan, Pushpa; Lenardo, Michael (2012) Comment on ""Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways"". J Immunol 188:5203-4; author reply 5204-5 |
| Snow, Andrew L; Xiao, Wenming; Stinson, Jeffrey R et al. (2012) Congenital B cell lymphocytosis explained by novel germline CARD11 mutations. J Exp Med 209:2247-61 |
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