Autoreactivity to Sulfatide by Human Invariant NKT Cells.

Invariant NKT (iNKT) cells are innate-like lymphocytes that recognize lipid Ags presented by CD1d. The prototypical Ag, α-galactosylceramide, strongly activates human and mouse iNKT cells, leading to the assumption that iNKT cell physiology in human and mouse is similar. In this article, we report the surprising finding that human, but not mouse, iNKT cells directly recognize myelin-derived sulfatide presented by CD1d. We propose that sulfatide is recognized only by human iNKT cells because of the unique positioning of the 3-O-sulfated ß-galactose headgroup. Surface plasmon resonance shows that the affinity of human CD1d-sulfatide for the iNKT cell receptor is relatively low compared with CD1d-α-galactosylceramide (KD of 19-26 µM versus 1 µM). Apolipoprotein E isolated from human cerebrospinal fluid carries sulfatide that can be captured by APCs and presented by CD1d to iNKT cells. APCs from patients with metachromatic leukodystrophy, who accumulate sulfatides due to a deficiency in arylsulfatase-A, directly activate iNKT cells. Thus, we have identified sulfatide as a self-lipid recognized by human iNKT cells and propose that sulfatide recognition by innate T cells may be an important pathologic feature of neuroinflammatory disease and that sulfatide in APCs may contribute to the endogenous pathway of iNKT cell activation.

Conjugation of A and B Blood Group Structures to Silica Microparticles for the Detection of Antigen-Specific B Cells.

Silica microparticles were functionalized with A and B blood group carbohydrate antigens (A type I, A type II, B type I, and B type II) to enable the detection and monitoring of ABO antigen-specific B cells. Microparticles were prepared via the Stöber synthesis, labeled with an Alexafluor fluorescent dye, and characterized via TEM and fluorescence microscopy. The silica microparticles were functionalized with (3-aminopropyl)trimethoxysilane (APTMS), followed by the use of an established fluorenylmethyloxycarbonyl (Fmoc)-protected PEG-based linker. The terminal Fmoc moiety of the PEG-based linker was then deprotected, yielding free amino groups, to which the A and B antigens were coupled. The carbohydrate antigens were synthesized with a p-nitrophenol ester to enable conjugation to the functionalized silica microparticles via an amide bond. The number of free amine groups available for coupling for a given mass of PEG-functionalized silica microparticles was quantified via reaction with Fmoc-glycine. The antigen-functionalized microparticles were then evaluated for their specificity in binding to A and B antigen-reactive B-cells via flow cytometry, and for blocking of naturally occurring antibodies in human serum. Selective binding of the functionalized microparticles to blood group-reactive B cells was observed by flow cytometry and fluorescence microscopy. The modular approach outlined here is applicable to the preparation of silica microparticles containing any carbohydrate antigen and alternative fluorophores or labels. This approach therefore comprises a novel, general platform for screening B cell populations for binding to carbohydrate antigens, including, in this case, the human A and B blood group antigens.

Characterization of pathogenic human monoclonal autoantibodies against GM-CSF.

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.

Innate immune control of EBV-infected B cells by invariant natural killer T cells.

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 EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α 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 γ 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.

CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling.

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 downregulation 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.

Apolipoprotein-mediated lipid antigen presentation in B cells provides a pathway for innate help by NKT cells.

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here, we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells, capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (alphaGalCer), an exogenous CD1d presented antigen, inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation, and the activation of NKT cells by B cells is completely LDL-R dependent, as shown by blocking experiments and the complete lack of presentation when using apoE2, an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells, which can apparently use alternate pathways of lipid antigen uptake. Thus, B cells use an apolipoprotein-mediated pathway of lipid antigen presentation, which constitutes a form of innate help for B cells by NKT cells.

High-throughput sequencing defines donor and recipient HLA B-cell epitope frequencies for prospective matching in transplantation.

Compatibility for human leukocyte antigen (HLA) genes between transplant donors and recipients improves graft survival but prospective matching is rarely performed due to the vast heterogeneity of this gene complex. To reduce complexity, we have combined next-generation sequencing and in silico mapping to determine transplant population frequencies and matching probabilities of 150 antibody-binding eplets across all 11 classical HLA genes in 2000 ethnically heterogeneous renal patients and donors. We show that eplets are more common and uniformly distributed between donors and recipients than the respective HLA isoforms. Simulations of targeted eplet matching shows that a high degree of overall compatibility, and perfect identity at the clinically important HLA class II loci, can be obtained within a patient waiting list of approximately 250 subjects. Internal epitope-based allocation is thus feasible for most major renal transplant programs, while regional or national sharing may be required for other solid organs.

Disruption of human plasma cell differentiation by an environmental polycyclic aromatic hydrocarbon: a mechanistic immunotoxicological study.

BACKGROUND: The AhR is a ligand-activated transcription factor that mediates immunosuppression induced by environmental PAH and HAH. Recently, a critical role for the AhR in development of T cells involved in autoimmunity (Th17 and Treg) has been demonstrated, supporting the hypothesis that the AhR plays a key role in immune regulation both in the presence and absence of environmental ligands. Despite these results with T cells systems, little is known of the role that the AhR plays in B cell development. We have demonstrated that B cell activation with CD40 ligand, a stimulus that models adaptive immunity, induces AhR expression in primary human B cells, suggesting that activation may increase human B cell sensitivity to AhR ligands and that the AhR may play a role in B cell development. METHODS: To test these possibilities, we developed an in vitro system in which activated human B cells expressing high AhR levels are induced to differentiate into plasma cells. Consequently, the effects of benzo [a]pyrene, a prototypic environmental AhR ligand, on plasma cell differentiation could be investigated and this chemical could be exploited essentially as drug probe to implicate the role of the AhR in plasma cell development. RESULTS: A previously unattainable level of B cell differentiation into plasma cells (up to 45% conversion) was observed. Benzo [a]pyrene significantly suppressed that differentiation. gamma-Irradiation after an initial proliferation phase induced by CD40 ligand and immediately prior to initiation of the differentiation phase blocked cell growth but did not affect cell viability or plasma cell differentiation. B [a]P suppressed differentiation whether or not cell growth was inhibited by gamma-irradiation. CONCLUSIONS: 1) Extensive proliferation is not required during the differentiation phase per se for CD40L-activated human B cells to undergo plasma cell differentiation, and 2) an environmental PAH blocks both proliferation and differentiation of AhR expressing B cells. The results uncover a new mechanism by which environmentally ubiquitous PAHs may negatively impact human B cell-mediated immunity.

Chronic lymphocytic leukemia patients with HLA-B27 referred for allogeneic hematopoietic stem cell transplantation do not have worse outcomes: Results of a population-based case series analysis in British Columbia, Canada.

Human leukocyte antigen B27 (HLA-B27), associated with spondyloarthritis, was suggested to be protective against chronic lymphocytic leukemia (CLL). It is hypothesized that HLA-B27 patients may have worse outcome in part related to their other comorbidities. OBJECTIVES: We sought to compare the clinical characteristics and outcomes of CLL and small lymphocytic lymphoma (SLL) patients referred for allogeneic hematopoietic stem cell transplantation (allo-HSCT) based on their HLA-B27 status. METHODS: This retrospective population-based case series analyzed CLL/SLL patients who were HLA-typed for potential allo-HSCT in British Columbia, Canada. RESULTS: of 279 CLL/SLL patients referred for potential allo-HSCT, 34 patients were HLA-B27 positive. For HLA-B27 patients, median age at CLL diagnosis was 53.5 years (range, 27-67) and 71% were male. Seven patients had 11q deletion and nine patients had 17p deletion detected prior to first CLL therapy or at relapse. Eleven HLA-B27 patients received allo-HSCT. Two patients developed acute myeloid leukemia. One patient with ankylosing spondylitis had Richter's transformation prior to any CLL therapy. Spondyloarthritis-related disorders were diagnosed in 12 HLA-B27 patients but there was no temporal correlation with development of CLL. Overall survival (OS) and treatment-free survival (TFS) were not significantly different between HLA-B27 patients with or without spondyloarthritis-related disorders. There were no significant differences in clinical characteristics at CLL diagnosis or OS/TFS between HLA-B27 positive and negative patients referred for allo-HSCT. CONCLUSIONS: HLA-B27 positivity does not appear to influence outcome for CLL/SLL patients referred for allo-HSCT. Further studies are needed to evaluate the clinical significance of HLA-B27 in a general CLL population.

Repression of aryl hydrocarbon receptor (AHR) signaling by AHR repressor: role of DNA binding and competition for AHR nuclear translocator.

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.

Bone marrow stromal-B cell interactions in polycyclic aromatic hydrocarbon-induced pro/pre-B cell apoptosis.

Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and alpha-naphthoflavone (alpha-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is > or = 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell-cell contact.

Regulation of constitutive and inducible AHR signaling: complex interactions involving the AHR repressor.

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 AHRR-mediated 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.

CYP1A1 in polycyclic aromatic hydrocarbon-induced B lymphocyte growth suppression.

The AhR is a ligand-activated transcription factor that mediates immunosuppression by environmental PAH. Previous studies demonstrated that activation of mature human B cells up-regulates AhR expression, suggesting that human B cells are direct PAH targets. To test this hypothesis and to determine the metabolic requirements for PAH toxicity in a human model, the effects of a prototypic PAH, B[a]P, on B cell growth were evaluated. B[a]P and its proximal (B[a]P-7,8-dihydrodiol) and terminal (B[a]P-7,8-dihydrodiol-9,10-epoxide) metabolites inhibited growth in a dose-dependent manner. A poorly metabolized AhR ligand had no effect, suggesting that biotransformation is required for growth inhibition. Inhibition of the CYP1A1 monooxygenase completely blocked growth inhibition induced by B[a]P or B[a]P-7,8-dihydrodiol, but not by B[a]P-dihydrodiol-9,10-epoxide, indicating that CYP1A1-dependent metabolism of B[a]P into the terminal B[a]P-7,8-dihydrodiol-9,10-epoxide metabolite is required for growth inhibition. These studies show for the first time the metabolic requirements for PAH-mediated suppression of human B cell growth.

Constitutive activation and environmental chemical induction of the aryl hydrocarbon receptor/transcription factor in activated human B lymphocytes.

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. Because B cells seem to be directly affected by AhR ligands in animal models, it was postulated that the AhR is predominantly expressed in activated human B cells and that it may contribute to cell growth regulation. To begin to address these issues and to extend detailed analyses of AhR function to a human system, AhR expression in resting and activated human B cells was studied. In addition, the response of activated B cells to an environmental AhR ligand was investigated to provide insight into a possible physiological role for the AhR. Resting peripheral human B cells expressed little or no AhR. However, activation with CpG or CD40 ligand profoundly up-regulated AhR mRNA and protein. AhR nuclear translocation, constitutive DNA binding, and induction of an AhR-regulated gene, CYP1A1, in stimulated B cells in the absence of exogenous ligands suggested constitutive AhR activation. Cell division was not required for AhR up-regulation. Treatment of AhR-expressing B cells with a prototypic environmental AhR ligand, benzo[a]pyrene, significantly suppressed cell growth. These data help explain the sensitivity of B cells to environmental AhR ligands and strongly suggest that the AhR plays an important function within the human B cell compartment.

Environmental chemical-induced bone marrow B cell apoptosis: death receptor-independent activation of a caspase-3 to caspase-8 pathway.

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 demonstrated previously with a nontransformed pro-/pre-B cell line, primary pro-B cells cultured on bone marrow stromal cells underwent apoptosis after exposure to a prototypic PAH, 7,12-dimethylbenz[a]anthracene (DMBA). Apoptosis was preceded by cleavage of caspase-3 (4-6 h) and caspase-8 (6-8 h) and their respective substrates, alpha-fodrin and Bid. Inhibition of caspase-3 blocked caspase-8 activation and apoptosis. Furthermore, a pan-caspase inhibitor blocked apoptosis and activation of both caspases-3 and -8. Cells from mice defective in tumor necrosis factor (TNF)-alpha, TNF-beta, lymphotoxin-beta, or TNFR1, TNFR2, Fas, or death receptor 6 were as susceptible to apoptosis signaling as wild-type cells. These results suggest a complex death receptor-independent B cell apoptosis pathway in which caspase-8 is activated downstream of caspase-3.