Harnessing regulatory T cells to establish immune tolerance.

Engineered regulatory T (Treg) cells have emerged as precision therapeutics aimed at inducing immune tolerance while reducing the risks associated with generalized immunosuppression. This Viewpoint highlights the opportunities and challenges for engineered Treg cell therapies in treating autoimmune and other inflammatory diseases.

Pharmacokinetic and Target Engagement Measures of ANX007, an Anti-C1q Antibody Fragment, Following Intravitreal Administration in Nonhuman Primates.

Purpose: C1q and the classical complement cascade are key regulators of synaptic pruning, and their aberrant activation has been implicated in neurodegenerative ophthalmic diseases including geographic atrophy and glaucoma. The antigen-binding fragment antibody ANX007 specifically recognizes globular head groups of C1q to block substrate binding and functionally inhibit classical complement cascade activation. ANX007 was assessed in nonclinical studies of biodistribution and C1q target engagement in the eye following intravitreal (IVT) administration in cynomolgus monkeys. Methods: Female juvenile cynomolgus monkeys (n = 12) received a single bilateral dose of 1 or 5 mg ANX007/eye, with vitreous and non-perfused tissue samples collected approximately 4 weeks later. In a separate study, male (n = 6/5) and female (n = 6/5) animals received repeat bilateral dosing of 1, 2.5, or 5 mg ANX007/eye on days 1 and 29, with aqueous and vitreous collections on day 44 or day 59. Tissues from the 5 mg/eye repeat-dose group were perfused, and retina, choroid, and optic nerve samples were collected approximately 2 and 4 weeks post-last dose. Results: Following a single dose of ANX007, vitreous levels of free drug were measurable through 4 weeks at both the 1 and 5 mg dose levels, with approximately 3-day half-life. With repeat dose of 5 mg/eye, free-ANX007 was measurable 4 weeks post-last dose in perfused retina and choroid and up to approximately 2 weeks post-last dose in optic nerve. There was a strong correlation between C1q target engagement and free drug levels in aqueous and vitreous humors and retinal tissue. Conclusions: Following IVT administration, ANX007 distributes to sites within the retina that are relevant to neurodegenerative ophthalmic disease with clear evidence of C1q target engagement. Based on its mechanism of action inhibiting C1q and its downstream activity, ANX007 is predicted to mitigate tissue damage driven by classical complement activation in the retina. These data support further clinical evaluation of ANX007.

Abnormalities in normal-appearing white matter from which multiple sclerosis lesions arise.

Normal-appearing white matter is far from normal in multiple sclerosis; little is known about the precise pathology or spatial pattern of this alteration and its relation to subsequent lesion formation. This study was undertaken to evaluate normal-appearing white matter abnormalities in brain areas where multiple sclerosis lesions subsequently form, and to investigate the spatial distribution of normal-appearing white matter abnormalities in persons with multiple sclerosis. Brain MRIs of pre-lesion normal-appearing white matter were analysed in participants with new T2 lesions, pooled from three clinical trials: SYNERGY (NCT01864148; n = 85 with relapsing multiple sclerosis) was the test data set; ASCEND (NCT01416181; n = 154 with secondary progressive multiple sclerosis) and ADVANCE (NCT00906399; n = 261 with relapsing-remitting multiple sclerosis) were used as validation data sets. Focal normal-appearing white matter tissue state was analysed prior to lesion formation in areas where new T2 lesions later formed (pre-lesion normal-appearing white matter) using normalized magnetization transfer ratio and T2-weighted (nT2) intensities, and compared with overall normal-appearing white matter and spatially matched contralateral normal-appearing white matter. Each outcome was analysed using linear mixed-effects models. Follow-up time (as a categorical variable), patient-level characteristics (including treatment group) and other baseline variables were treated as fixed effects. In SYNERGY, nT2 intensity was significantly higher, and normalized magnetization transfer ratio was lower in pre-lesion normal-appearing white matter versus overall and contralateral normal-appearing white matter at all time points up to 24 weeks before new T2 lesion onset. In ASCEND and ADVANCE (for which normalized magnetization transfer ratio was not available), nT2 intensity in pre-lesion normal-appearing white matter was significantly higher compared to both overall and contralateral normal-appearing white matter at all pre-lesion time points extending up to 2 years prior to lesion formation. In all trials, nT2 intensity in the contralateral normal-appearing white matter was also significantly higher at all pre-lesion time points compared to overall normal-appearing white matter. Brain atlases of normal-appearing white matter abnormalities were generated using measures of voxel-wise differences in normalized magnetization transfer ratio of normal-appearing white matter in persons with multiple sclerosis compared to scanner-matched healthy controls. We observed that overall spatial distribution of normal-appearing white matter abnormalities in persons with multiple sclerosis largely recapitulated the anatomical distribution of probabilities of T2 hyperintense lesions. Overall, these findings suggest that intrinsic spatial properties and/or longstanding precursory abnormalities of normal-appearing white matter tissue may contribute to the risk of autoimmune acute demyelination in multiple sclerosis.

MOG autoantibodies trigger a tightly-controlled FcR and BTK-driven microglia proliferative response.

Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease.

Cell-autonomous and non-cell autonomous effects of neuronal BIN1 loss in vivo.

BIN1 is the most important risk locus for Late Onset Alzheimer's Disease (LOAD), after ApoE. BIN1 AD-associated SNPs correlate with Tau deposition as well as with brain atrophy. Furthermore, the level of neuronal-specific BIN1 isoform 1 protein is decreased in sporadic AD cases in parallel with neuronal loss, despite an overall increase in BIN1 total mRNA. To address the relationship between reduction of BIN1 and neuronal cell loss in the context of Tau pathology, we knocked-down endogenous murine Bin1 via stereotaxic injection of AAV-Bin1 shRNA in the hippocampus of mice expressing Tau P301S (PS19). We observed a statistically significant reduction in the number of neurons in the hippocampus of mice injected with AAV-Bin1 shRNA in comparison with mice injected with AAV control. To investigate whether neuronal loss is due to deletion of Bin1 selectively in neurons in presence Tau P301S, we bred Bin1flox/flox with Thy1-Cre and subsequently with PS19 mice. Mice lacking neuronal Bin1 and expressing Tau P301S showed increased mortality, without increased neuropathology, when compared to neuronal Bin1 and Tau P301S-expressing mice. The loss of Bin1 isoform 1 resulted in reduced excitability in primary neurons in vitro, reduced neuronal c-fos expression as well as in altered microglia transcriptome in vivo. Taken together, our data suggest that the contribution of genetic variation in BIN1 locus to AD risk could result from a cell-autonomous reduction of neuronal excitability due to Bin1 decrease, exacerbated by the presence of aggregated Tau, coupled with a non-cell autonomous microglia activation.

BIN1 favors the spreading of Tau via extracellular vesicles.

Despite Bridging INtegrator 1 (BIN1) being the second most statistically-significant locus associated to Late Onset Alzheimer's Disease, its role in disease pathogenesis remains to be clarified. As reports suggest a link between BIN1, Tau and extracellular vesicles, we investigated whether BIN1 could affect Tau spreading via exosomes secretion. We observed that BIN1-associated Tau-containing extracellular vesicles purified from cerebrospinal fluid of AD-affected individuals are seeding-competent. We showed that BIN1 over-expression promotes the release of Tau via extracellular vesicles in vitro as well as exacerbation of Tau pathology in vivo in PS19 mice. Genetic deletion of Bin1 from microglia resulted in reduction of Tau secretion via extracellular vesicles in vitro, and in decrease of Tau spreading in vivo in male, but not female, mice, in the context of PS19 background. Interestingly, ablation of Bin1 in microglia of male mice resulted in significant reduction in the expression of heat-shock proteins, previously implicated in Tau proteostasis. These observations suggest that BIN1 could contribute to the progression of AD-related Tau pathology by altering Tau clearance and promoting release of Tau-enriched extracellular vesicles by microglia.

Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation.

Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism.

Identification of regulators of the myofibroblast phenotype of primary dermal fibroblasts from early diffuse systemic sclerosis patients.

Systemic sclerosis (SSc or scleroderma) is an auto-immune disease characterized by skin fibrosis. While primary cells from patients are considered as a unique resource to better understand human disease biology, the effect of in vitro culture on these cells and their evaluation as a platform to identify disease regulators remain poorly characterized. The goal of our studies was to provide insights into the utility of SSc dermal fibroblast primary cells for therapeutic target discovery. The disease phenotypes of freshly isolated and in vitro cultured SSc dermal fibroblasts were characterized using whole transcriptome profiling, alpha smooth muscle actin (ASMA) expression and cell impedance. SSc dermal fibroblasts retained most of the molecular disease phenotype upon in vitro culture for at least four cell culture passages (approximatively 10 cell doublings). We validated an RNA interference high throughput assay that successfully identified genes affecting the myofibroblast phenotype of SSc skin fibroblasts. These genes included MKL1, RHOA and LOXL2 that were previously proposed as therapeutic anti-fibrotic target, and ITGA5, that has been less studied in fibrosis biology and may be a novel potential modifier of SSc fibroblast biology. Together our results demonstrated the value of carefully-phenotyped SSc dermal fibroblasts as a platform for SSc target and drug discovery.

Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism.

APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells.IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5'-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.

CDK12-mediated transcriptional regulation of noncanonical NF-κB components is essential for signaling.

Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.

A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization.

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Infectious Entry and Neutralization of Pathogenic JC Polyomaviruses.

Progressive multifocal leukoencephalopathy (PML) is a lethal brain disease caused by uncontrolled replication of JC polyomavirus (JCV). JCV strains recovered from the brains of PML patients carry mutations that prevent the engagement of sialylated glycans, which are thought to serve as receptors for the infectious entry of wild-type JCV. In this report, we show that non-sialylated glycosaminoglycans (GAGs) can serve as alternative attachment receptors for the infectious entry of both wild-type and PML mutant JCV strains. After GAG-mediated attachment, PML mutant strains engage non-sialylated non-GAG co-receptor glycans, such as asialo-GM1. JCV-neutralizing monoclonal antibodies isolated from patients who recovered from PML appear to block infection by preventing the docking of post-attachment co-receptor glycans in an apical pocket of the JCV major capsid protein. Identification of the GAG-dependent/sialylated glycan-independent alternative entry pathway should facilitate the development of infection inhibitors, including recombinant neutralizing antibodies.

Distribution and Quantity of Sites of John Cunningham Virus Persistence in Immunologically Healthy Patients: Correlation With John Cunningham Virus Antibody and Urine John Cunningham Virus DNA.

Importance: Although seroepidemiological studies indicate that greater than 50% of the population has been infected with John Cunningham virus (JCV), the sites of JCV persistence remain incompletely characterized. Objective: To determine sites of JCV persistence in immunologically healthy individuals. Design, Setting, and Participants: Tissue specimens from multiple sites including brain, renal, and nonrenal tissues were obtained at autopsy performed in the Department of Pathology at the University of Kentucky from 12 immunologically healthy patients between February 9, 2011, and November 27, 2012. Quantitative polymerase chain reaction was performed on the tissue specimens and urine. Serum JCV antibody status was determined by enzyme-linked immunosorbent assay. Main Outcomes and Measures: The detection and quantification of JCV from the tissues by quantitative polymerase chain reaction illuminated sites of viral persistence. These results were correlated with JCV antibody levels. Results: Autopsies were performed on 12 individuals, 10 men and 2 women, ranging in age from 25 to 75 years (mean, 55.3 years). Seven of 12 individuals were JCV antibody seropositive based on absorbance units. Serostatus was associated with amounts of JCV DNA in urine and its tissue distribution. John Cunningham virus DNA was found in 75% of genitourinary tissue samples from donors (18 of 24) with high JCV antibody levels, 13.3% of donors with low levels i(4 of 30), and 0% of seronegative persons (0 of 32). In nongenitourinary tissues, JCV DNA was detected in 45.1% of tissue samples of donors (32 of 71) with high JCV, 2.2% of donors with low JCV serostatus (2 of 93), and 0% of seronegative persons (0 of 43). Genitourinary tissues had higher copy numbers than other sites. John Cunningham virus DNA was detected in urine of seronegative individuals in a research-grade assay. Conclusions and Relevance: Persistent (latent or actively replicating) JCV infection mostly predominates in genitourinary tissues but distributes in other tissues at low copy number. The distribution and copy numbers of the virus appear to correlate with urinary JCV shedding and serostatus.

Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9.

Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV). Currently, there is no anti-viral therapeutic available against JCPyV infection. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) is a genome editing tool capable of introducing sequence specific breaks in double stranded DNA. Here we show that the CRISPR/Cas9 system can restrict the JCPyV life cycle in cultured cells. We utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. We found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.

JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection.

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.

CD62L is not a reliable biomarker for predicting PML risk in natalizumab-treated R-MS patients.

OBJECTIVE: To assess if the percentage of CD3(+)CD4(+)CD62L(+) cells in cryopreserved peripheral blood mononuclear cells (PBMCs) (here termed %CD62L) can predict risk of developing progressive multifocal leukoencephalopathy (PML) and better inform the physician for benefit-risk assessment of natalizumab treatment decisions in a global setting. METHODS: Cryopreserved PBMCs from 21 natalizumab-treated patients who developed PML and 104 matched natalizumab-treated patients with multiple sclerosis (MS) without PML collected as a part of Biogen clinical trials were retrospectively examined for CD3, CD4, CCR7, CD45RA, and CD62L by flow cytometry. RESULTS: In this cohort, %CD62L in natalizumab-treated patients did not predict PML risk. Natalizumab-treated patients with MS without PML showed highly variable %CD62L upon serial sampling. In the STRATA study, the distribution of %CD62L in samples collected more than 6 months before a PML diagnosis, at diagnosis, and in natalizumab-treated patients without PML overlapped. No statistical threshold for risk could be determined. In addition, we demonstrated that lymphocyte viability strongly affects %CD62L, supporting previous reports that %CD62L is inherently unstable following cryopreservation and is sensitive to sample collection. CONCLUSION: Data from this well-controlled cohort of natalizumab-treated patients indicate that %CD62L is not a biomarker of PML risk.

The NF-κB genomic landscape in lymphoblastoid B cells.

The nuclear factor κB (NF-κΒ) subunits RelA, RelB, cRel, p50, and p52 are each critical for B cell development and function. To systematically characterize their responses to canonical and noncanonical NF-κB pathway activity, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis in lymphoblastoid B cell lines (LCLs). We found a complex NF-κB-binding landscape, which did not readily reflect the two NF-κB pathway paradigms. Instead, 10 subunit-binding patterns were observed at promoters and 11 at enhancers. Nearly one-third of NF-κB-binding sites lacked κB motifs and were instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupied nearly half of NF-κB-binding sites and was identified in protein complexes with NF-κB on DNA. FOXM1 knockdown decreased NF-κB target gene expression and ultimately induced apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy. These studies provide a resource for understanding mechanisms that underlie NF-κB nuclear activity and highlight opportunities for selective NF-κB blockade.

Pharmacogenomics: novel loci identification via integrating gene differential analysis and eQTL analysis.

Nearly one-half of asthmatic patients do not respond to the most commonly prescribed controller therapy, inhaled corticosteroids (ICS). We conducted an expression quantitative trait loci (eQTL) analysis using >300 expression microarrays (from 117 lymphoblastoid cell lines) in corticosteroid (dexamethasone) treated and untreated cells derived from asthmatic subjects in the Childhood Asthma Management Program (CAMP) clinical trial. We then tested the associations of eQTL with longitudinal change in airway responsiveness to methacholine (LnPC20) on ICS. We identified 2484 cis-eQTL affecting 767 genes following dexamethasone treatment. A significant over-representation of lnPC20-associated cis-eQTL [190 single-nucleotide polymorphisms (SNPs)] among differentially expressed genes (odds ratio = 1.76, 95% confidence interval: 1.35-2.29) was noted in CAMP Caucasians. Forty-six of these 190 clinical associations were replicated in CAMP African Americans, including seven SNPs near six genes meeting criteria for genome-wide significance (P < 2 × 10(-7)). Notably, the majority of genome-wide findings would not have been uncovered via analysis of untreated samples. These results indicate that identifying eQTL after relevant environmental perturbation enables identification of true pharmacogenetic variants.