Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin causes altered gene expression and toxicity. The AHR repressor (AHRR) inhibits AHR signaling through a proposed mechanism involving competition with AHR for dimerization with AHR nuclear translocator (ARNT) and binding to AHR-responsive enhancer elements (AHREs). We sought to delineate the relative roles of competition for ARNT and AHREs in the mechanism of repression. In transient transfections in which AHR2-dependent transactivation was repressed by AHRR1 or AHRR2, increasing ARNT expression failed to reverse the repression, suggesting that AHRR inhibition of AHR signaling does not occur through sequestration of ARNT. An AHRR1 point mutant (AHRR1-Y9F) that could not bind to AHREs but that retained its nuclear localization was only slightly reduced in its ability to repress AHR2, demonstrating that AHRR repression does not occur solely through competition for AHREs. When both proposed mechanisms were blocked (AHRR1-Y9F plus excess ARNT), AHRR remained functional. AHRR1 neither blocked AHR nuclear translocation nor reduced the levels of AHR2 protein. Experiments using AHRR1 C-terminal deletion mutants showed that amino acids 270 to 550 are dispensable for repression. These results demonstrate that repression of AHR transactivation by AHRR involves the N-terminal portion of AHRR; does not involve competition for ARNT; and does not require binding to AHREs, although AHRE binding can contribute to the repression. We propose a mechanism of AHRR action involving "transrepression" of AHR signaling through protein-protein interactions rather than by inhibition of the formation or DNA binding of the AHR-ARNT complex.

The AHR is well known for regulating responses to an array of environmental chemicals. A growing body of evidence supports the hypothesis that the AHR also plays perhaps an even more important role in modulating critical aspects of cell function including cell growth, death, and migration. As these and other important AHR activities continue to be elucidated, it becomes apparent that attention now must be directed towards the mechanisms through which the AHR itself is regulated. Here, we review what is known of and what biological outcomes have been attributed to the AHR repressor (AHRR), an evolutionarily conserved bHLH-PAS protein that inhibits both xenobiotic-induced and constitutively active AHR transcriptional activity in multiple species. We discuss the structure and evolution of the AHRR and the dominant paradigm of a xenobiotic-inducible negative feedback loop comprised of AHR-mediated transcriptional up-regulation of AHRR and the subsequent AHRRmediated suppression of AHR activity. We highlight the role of the AHRR in limiting AHR activity in the absence of xenobiotic AHR ligands and the important contribution of constitutively repressive AHRR to cancer biology. In this context, we also suggest a new hypothesis proposing that, under some circumstances, constitutively active AHR may repress AHRR transcription, resulting in unbridled AHR activity. We also review the predominant hypotheses on the molecular mechanisms through which AHRR inhibits AHR as well as novel mechanisms through which the AHRR may exert AHR-independent effects. Collectively, this discussion emphasizes the importance of this understudied bHLH-PAS protein in tissue development, normal cell biology, xenobiotic responsiveness, and AHR-regulated malignancy.

Key Points• B cells rapidly downregulate CD1d expression after EBV infection, thus abrogating iNKT cell recognition.• EBV-infected B cells induced to express CD1d elicit iNKT cell functions even in the absence of exogenous antigen.Individuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBVinfected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-a agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon g secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition. (Blood. 2013;122(15):2600-2608

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates immunosuppression induced by a variety of ubiquitous environmental pollutants, including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and dioxins. Although the normal physiological role for the AhR in the absence of environmental chemicals is uncertain, recent studies suggest its contribution to cell growth and apoptosis.

IntroductionUnlike peptide antigens, which are presented to conventional T cells via major histocompatibility complex (MHC) molecules, lipid antigens are presented to T cells by the MHC-like molecule, CD1. Humans express several nonpolymorphic CD1 molecules, including CD1d, which presents lipids to a unique subset of T cells termed natural killer T (NKT) cells. NKT cells are innate-like lymphocytes defined by their characteristic semi-invariant T-cell receptor that recognizes the potent glycolipid antigen ␣-galactosylceramide (␣GalCer). In addition to this nonphysiologic antigen, NKT cells have recently been shown to respond to exogenous bacterial-derived lipid antigens 1-3 as well as endogenous lipids presented by antigen-presenting cells (APCs) responding to innate stimuli. 2,4,5 Resulting in part from their inherent memory phenotype and rapid cytokine secretion after stimulation, NKT cells complement innate signals and promote adaptive immunity. 6 ␣GalCer has been shown to have adjuvant-like properties that enhance T-dependent and T-independent humoral immune responses. [7][8][9][10] To generate antibodies to T-dependent antigens, B cells require cognate T-cell help from conventional T helper (Th) cells that recognize the same antigen or antigenic complex, which has been internalized by the B-cell receptor (BCR). ␣GalCer has been shown to boost dendritic cell (DC)-Th interactions that augment Th priming and indirectly promote B-cell help. 11 However, B cells also express CD1d and have been shown to present lipid antigens and recruit cognate help from NKT cells. 8,9,12,13 Recent work has shown that BCR-mediated uptake of model B-cell antigens linked to ␣GalCer elicits cognate NKT help for lipid-specific B cell responses in vivo and in vitro. [12][13][14] However, B cells also internalize and present lipid antigens to NKT cells by pathways that are independent of the BCR, 15 and the mechanisms by which this occurs are unknown. Our previous studies have shown that DCs use the low-density lipoprotein (LDL)-receptor pathway to endocytose apolipoprotein E (apoE)-bound lipid antigens for subsequent presentation to NKT cells. 16 ApoE, found in serum very low density lipoproteins or secreted locally by DCs and macrophages, rapidly binds exogenous lipid antigens. ApoE-lipid antigen complexes are efficiently captured by the LDL-R on DCs and delivered to intracellular CD1d where the lipid antigen is loaded. Whereas DCs use multiple nonspecific pathways of antigen uptake, B-cell antigen uptake is thought to be restricted to the BCR. An LDL-R-mediated pathway in B cells would provide a means for innate help by NKT cells stimulating polyclonal B-cell activation. We thus investigated whether B cells may also use an apoEmediated pathway for lipid antigen presentation. Methods NKT cellsTwo CD1d-restricted human NKT cell clones (BM2a.3 and J3N.5, previously described 17 ) and 1 CD1d-restricted human NKT line (M0) were used in these studies, and similar results were obtained. M0 was derived by successive rounds of ␣GalCer-CD1d-t...

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downreglation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.

Programmed cell death is a critical process in B lymphocyte development. Premature apoptosis in developing B cells could affect the repertoire and number of mature B cells produced. Of particular concern is the ability of environmentally ubiquitous polycyclic aromatic hydrocarbons (PAH) to induce B cell apoptosis within the bone marrow microenvironment in a clonally nonspecific way. Here, models of bone marrow B cell development were used to assess the role of the "extrinsic" apoptosis pathway in PAH-induced apoptosis and to compare PAH-induced apoptosis with that induced during clonal deletion. As

The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain.