Kari C. Nadeau

Personal Omics Profiling Reveals Dynamic Molecular and Medical Phenotypes

Personalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here, we present an integrative personal omics profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14 month period. Our iPOP analysis revealed various medical risks, including type 2 diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high-coverage genomic and transcriptomic data, which provide the basis of our iPOP, revealed extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and diseased states by connecting genomic information with additional dynamic omics activity.

The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand.

Ischemia/reperfusion (I/R) injury associated with renal transplantation may influence both early graft function and late changes. The initial (</= 7 d) events of warm and in situ perfused cold ischemia of native kidneys in uninephrectomized rats were examined. mRNA expression of the early adhesion molecule, E-selectin, peaked within 6 h; PMNs infiltrated in parallel. T cells and macrophages entered the injured kidney by 2-5 d; the associated upregulation of MHC class II antigen expression suggested increased immunogenicity of the organ. Th1 products (IL-2, TNFalpha, IFNgamma) and macrophage-associated products (IL-1, IL-6, TGFbeta) remained highly expressed after 2 d. To examine directly the effects of selectins in I/R injury, a soluble P-selectin glycoprotein ligand (sPSGL) was used. Ischemic kidneys were perfused in situ with 5 microg of sPSGL in UW solution; 50 microg was administered intravenously 3 h after reperfusion. E-selectin mRNA remained at baseline, leukocytes did not infiltrate the injured organs throughout the 7-d period, and their associated products were markedly inhibited. Class II expression did not increase. No renal dysfunction secondary to I/R occurred. The early changes of I/R injury may be prevented by treatment with soluble P- and E-selectin ligand. This may reduce subsequent host inflammatory responses after transplantation.

Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing

Massively parallel sequencing of rearranged immune receptor genes permits detection and tracking of specific immune cell populations in normal and pathological contexts. Like a reporter who serially unearths fragments of a story until a plausible picture of the latest scandal emerges, scientists have over time gathered pieces of the vast amount of information inherent in the highly recombined genes of the human immune system—probing their complexity, seeking a disease diagnosis, or hunting for evidence of remission. Back in 1987, Susumu Tonegawa won the Nobel Prize in Physiology or Medicine for discovering the genetics behind the diversity of human antibodies—a process called V-D-J recombination. Now, more than 20 years later, scientists at Stanford University and 454 Life Sciences have used powerful next-generation DNA sequencing technology to comprehensively characterize the products of V-D-J recombination in both cancer patients and healthy volunteers. Indeed, this ability to exhaustively profile the human immune response will help to untangle some of biomedicine’s most knotty problems—cancer, autoimmune disease, and vaccine development. B and T lymphocytes, cells of the adaptive immune system, build the blueprints for myriad antigen-recognizing proteins—immunoglobulins (Ig) and T cell receptors—by recombination within variable (V), diversity (D), and joining (J) gene segments to rearrange the intervening highly variable DNA sequences that can specify numerous antigen recognition domains. All of this reassortment creates a repertoire of receptors that recognizes scads of molecules from foreign invaders (antigens), a process that spurs the immune system to respond to the threat. When an immune cell sporting a particular antigen receptor finds and binds its matching antigen, the cell divides repeatedly, giving rise to many genetically identical lymphocytes that target a particular antigen for elimination. In contrast to this vibrant diversity of healthy immune systems, those of people with B lymphocyte– or T lymphocyte–based cancers (lymphomas or leukemias) generate cells that express a single dominant (clonal) receptor. In the new work, Boyd et al. performed massively parallel DNA sequencing of rearranged IgH gene loci in blood and tissue samples from cancer patients and healthy people to examine the diversity of their B cells, the immune cells that make antibodies. To this end, they amplified the rearranged IgH B cell DNA with a series of primers and the polymerase chain reaction to generate bar-coded, amplified DNA mixtures. These samples were then sequenced and the information was analyzed to determine which DNA segments had been joined to generate the blueprints for the IgH immune molecules. The experimental design used by Boyd et al. employs a high-throughput deep sequencing machine and can accommodate up to 150 samples at a time, providing an intricate snapshot of the immune repertoire. From healthy individuals, the authors were able to estimate the normal complexity of the B cell repertoire. With samples from the cancer patients, they obtained disease-specific signatures of clonal B cell proliferation events. For example, in a lymph node sample from one patient, deep sequencing detected two distinct V-D-J rearrangements. This finding indicates that there were two separate clonal B cell populations in this specimen and, therefore, two different B cell lymphomas. Such signatures could be obtained at the time of disease diagnosis and then monitored on an ongoing basis and thereby used to assess the effects of anticancer therapies that target these clonal populations or for early detection of disease relapse. Characterization of immune cell populations by deep sequencing also may illuminate fundamental aspects of infectious and autoimmune diseases as well as the body’s response to vaccination, gene and cell therapies, and other surgical procedures. The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. Here, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of bar-coded amplicons were sequenced with long-read ultradeep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or reemergence of such populations after treatment.

Rapid oral desensitization in combination with omalizumab therapy in patients with cow’s milk allergy

To the Editor: We conducted a pilot phase I study in 11 children (age, 7-17 years) with cow’s milk allergy by using omalizumab (anti-IgE mAb; Xolair; Genentech, South San Francisco, Calif) in combination with relatively rapid oral milk desensitization. We hypothesized that oral desensitization might occur rapidly and with few side effects when performed with omalizumab. Our primary objectives were to examine the safety of this approach and to determine whether subjects could be dosed up to 2000 mg milk within 7 to 11 weeks of initiating the desensitization. Eleven patients with a history of IgE-mediated milk allergy were enrolled in the study at 2 sites—Children’s Hospital Boston and Stanford University—under institutional review board and US Food and Drug Administration approval. All subjects had histories of acute clinical reactions to milk, including immediate reactions (urticaria, vomiting, and/or anaphylaxis) after ingestion of milk, as well as elevated milk-specific IgE (median, 50 kilounits of antibody (kUA)/L; range, 41.6-342 kUA/L; Table I). At entry, the median wheal/flare skin prick test to milk was 20/50 mm (wheal/erythema diameter; range, 11-45/20-52 mm), and the median total serum IgE was 349 kU/L (range, 148-2593 kU/L). The median age was 8 years (range, 7-17 years). Seven subjects had a diagnosis of asthma and/or eczema. For children with IgE levels 700 kU/L, the dose was 225 to 300 mg (approximately 0.016 mg/kg/IgE [U/ mL]) every 2 to 4 weeks. During the course of the study, subjects were asked to exclude all dairy products from their diets except what was given as the study milk dose. TABLE I Characteristics of enrolled subjects Nine weeks after the start of omalizumab treatment, oral cow’s milk desensitization was performed in 2 phases. Rush oral desensitization occurred on the first day of desensitization, starting with 0.1 mg of milk powder (dried nonfat powdered cow’s milk, Carnation Instant Milk; Nestle, San Francisco, Calif), with doses every 30 minutes to a maximum dose of 1000 mg (cumulative dose, 1992 mg). One subject (subject 7; Table I) voluntarily discontinued the study because of abdominal migraines; eosinophilic esophagitis and other allergic disorders were ruled out. Nine of the 10 remaining subjects reached the 1000-mg dose on the first day of desensitization. However, 1 subject, subject 5 (Table I), after administration of the 1000-mg dose, received epinephrine for nasal obstruction and generalized urticaria refractory to diphenhydramine and cetirizine. Subject 8 reacted at the 7-mg dose on the first day. Desensitization with daily doses of milk continued in the 10 subjects, with weekly increases in the dose of milk over the next 7 to 11 weeks (all dose increases were given in the clinical research unit, and if the dose was tolerated, the dose was then given daily at home). During the study, subjects were asked to take the study milk dose on a full stomach and to half the study milk dose during a viral infection. Nine of the 10 patients reached the maximum daily dose of 2000 mg milk (the primary end point of the study); the subject who received epinephrine during the rush phase of desensitization achieved a daily dose of 1200 mg when the omalizumab was stopped (end of the weekly dose escalation phase, week 16). Omalizumab treatment was then discontinued at week 16, whereas daily oral milk was continued at home. A double-blind, placebo-controlled food challenge (DBPCFC) was performed 8 weeks later (week 24 of the study). The DBPCFC consisted of 5 doses (milk or placebo, eg, rice or soy beverage) administered orally every 15 minutes: 500 mg, 750 mg, 1000 mg, 2000 mg, and 3000 mg (cumulative dose, 7250 mg, equivalent to 220 mL milk). Allergic reactions occurring during the protocol were scored by using the system developed by Bock et al.1 All 9 patients who had reached a daily dose of 2000 mg passed the DBPCFC and an open challenge (for subjects taking their oral food challenge on the same day of the DBPCFC [n = 4], 4000 mg was given as the open challenge; for subjects taking their open challenge on the day after the DBPCFC [n = 5], 8000 mg was given). All 9 patients continued with daily milk ingestion >8000 mg/d, which included different types of milk products. In terms of overall safety, the mean frequency for total reactions reported by week 24 was 1.6% (32 reactions of 2199 doses total for all 11 subjects; Table II). All patients experienced some adverse events, though most reactions were defined as mild1 (1%) and needed no treatment. There were moderate reactions (0.3%), and these included abdominal pain and vomiting, which responded within 1 hour to oral antihistamine dosing. Severe reactions (0.1%) included swelling of the tongue in 1 subject during the initial rush desensitization day, which responded to oral antihistamines. Another subject developed rhinitis and urticaria after the 1000-mg dose during the rush desensitization and responded to epinephrine. The most common types of reactions were local (mostly pruritus or urticaria) and/ or gastrointestinal (eg, abdominal pain), occurring with a frequency of 1%. No reactions occurred that involved the cardiovascular system or that failed to respond rapidly to treatment. There were 2 other subjects who were given epinephrine at home by their guardians during the maintenance phase of dosing. One received epinephrine for a moderate reaction that was manifested by upper lip swelling and urticaria (7.5 cm × 5cm) on the left upper leg; the second received epinephrine for urticaria (2.5 cm × 5 cm) on the right upper arm. In addition, this subject had wheezing, which began before the milk ingestion that day, and which most likely was a result of a viral infection. Overall, the reaction rate in our study was relatively low given the rapidity of the desensitization, although the rate of epinephrine use was similar to that in previous desensitization studies. All subjects tolerated omalizumab treatment with no signs of allergic reactions. TABLE II Overall safety data Previous studies of slow, deliberate oral milk desensitization in patients with cow’s milk allergy showed that desensitization can increase the amount of milk tolerated by many of the treated subjects.2-6 We now show that milk desensitization can be performed relatively rapidly with minimal hospitalization time when combined with omalizumab treatment. The limitations of our study include the small sample size, the lack of a placebo group, and lack of a baseline oral food challenge. In addition, it is possible that our desensitization protocol with omalizumab might be further optimized by limiting the number of milk doses during the rush phase; by extending the dose escalation phase over a longer period, beyond the 7 to 11 weeks used in our current protocol; and by performing the DBPCFC 12 or more weeks after discontinuation of omalizumab. In summary, we demonstrated that omalizumab treatment combined with oral milk desensitization in children with clinical reactions to cow’s milk permitted rapid milk dose escalation in the majority of subjects. This study is the first to use omalizumab in combination with oral desensitization and demonstrates a potential value of this approach for the treatment of food allergy, a major public health problem,7-9 although it must be first confirmed by future phase II and III trials. Nine of the 11 patients achieved the primary objective, tolerating desensitization to a dose of 2000 mg/d within a period of 7 to 11 weeks. Moreover, 9 of the 10 patients who completed the study passed a DBPCFC and an open challenge of milk without symptoms. Importantly, the 9 patients, after passing the DBPCFC, began tolerating almost normal amounts of milk in their diet (≥240 mL, equivalent to ≥8000 mg/d). The tenth patient is tolerating 4000 mg/d.

Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3)

BACKGROUND The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance. OBJECTIVE Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy. METHODS In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13). RESULTS Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells. CONCLUSION In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.

The role of the B7 costimulatory pathway in experimental cold ischemia/reperfusion injury.

Ischemia/reperfusion injury associated with organ retrieval and storage influences the development of chronic graft dysfunction, the major clinical problem in solid organ transplantation. The potential role of mononuclear cells (T cells and monocyte/macrophages) in this type of injury is unknown. Inbred male Lewis rats were uninephrectomized and the left kidney perfused in situ with 10 ml of iced University of Wisconsin solution. Immunohistological studies showed mononuclear cell infiltration of the ischemic organs associated with the upregulation of MHC class II antigen expression. Reverse transcriptase-PCR indicated that T cell associated cytokines and monocyte/macrophage activation markers/products are upregulated early after the ischemic insult. B7 expression occurred within 24 h and peaked at 3 d. Plasma creatinine levels rose transiently with complete recovery of renal function by 5 d. Animals began to develop progressive proteinuria after 8-12 wk, indicative of the long-term functional consequences of early ischemia/reperfusion injury. Blockade of T cell CD28-B7 costimulation with CTLA4Ig resulted in significant inhibition of T cell and macrophage infiltration and activation in situ. Treated animals did not exhibit transient renal dysfunction, nor developed proteinuria over time. This is the first demonstration that blocking T cell costimulatory activation in the absence of alloantigen can prevent the early and late consequences of ischemia/reperfusion injury.

Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individuals Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

Cutting Edge: Decreased Accumulation and Regulatory Function of CD4+CD25high T Cells in Human STAT5b Deficiency

We show that STAT5b is important for the in vivo accumulation of CD4+CD25high T cells with regulatory cell function. A patient homozygous for a missense A630P STAT5b mutation displayed immune dysregulation and decreased numbers of CD4+CD25high T cells. STAT5bA630P/A630P CD4+CD25high T cells had low expression of forkhead box P3 and an impaired ability to suppress the proliferation of or to kill CD4+CD25− T cells. Expression of CD25, a component of the high-affinity IL-2R, was also reduced in response to IL-2 or after in vitro propagation. The impact of the STAT5b mutation was selective in that IL-2-mediated up-regulation of the common γ-chain cytokine receptor and perforin, and activation-induced expressions of CD154 and IFN-γ were normal. These results indicate that STAT5b propagates an important IL-2-mediated signal for the in vivo accumulation of functional regulatory T cells.

Intravenous anti–IL-13 mAb QAX576 for the treatment of eosinophilic esophagitis

BACKGROUND Eosinophilic esophagitis (EoE) is a chronic allergic disease with limited treatment options. OBJECTIVE We evaluated QAX576, an mAb against IL-13, in the treatment of patients with EoE. METHODS Patients (18-50 years) with proton pump inhibitor-resistant esophageal eosinophilia received intravenous QAX576 (6 mg/kg) or placebo (2:1) at weeks 0, 4, and 8 and were followed for 6 months. The primary end point was the responder rate for a greater than 75% decrease in peak eosinophil counts at week 12. Efficacy was to be declared if the lower 90% confidence limit for the proportion of responders on QAX576 was 35% or greater. Secondary end points included changes in esophageal eosinophil counts, symptoms assessed by questionnaire scores, and quantification of a series of biomarkers. RESULTS Twenty-three patients completed the study up to week 12, and 18 continued to the end of the study. For the proximal and distal esophageal biopsies combined, the responder rate was 12.5% (90% confidence limit, 1% to 43%) with placebo, compared to 40.0% (90% confidence limit, 22% to 61%) with QAX576. Although the primary end point was not met, the mean esophageal eosinophil count decreased by 60% with QAX576 versus an increase of 23% with placebo (P = .004), and the decrease was sustained up to 6 months. There was a trend for improved symptoms, particularly dysphagia. QAX576 improved expression of EoE-relevant esophageal transcripts, including eotaxin-3, periostin, and markers of mast cells and barrier function, for up to 6 months after treatment. QAX576 was well tolerated. CONCLUSIONS QAX576 significantly improved intraepithelial esophageal eosinophil counts and dysregulated esophageal disease-related transcripts in adults with EoE in a sustained manner.

Epigenetic modifications and improved regulatory T-cell function in subjects undergoing dual sublingual immunotherapy

BACKGROUND Allergen-specific immunotherapy is the only mode of therapy that has been demonstrated to offer a cure in patients with IgE-mediated respiratory allergies. OBJECTIVE We sought to demonstrate the safety and efficacy of timothy grass (TG) and dust mite (DM) dual sublingual immunotherapy (SLIT) and to begin to investigate the immune mechanisms involved in successful immunotherapy with multiple allergens. METHODS The safety and efficacy of dual SLIT with TG and DM in children and adults with demonstrated allergies to TG and DM were investigated in a single-center, randomized, double-blind, controlled phase I study. Thirty subjects received either TG and DM dual SLIT (n= 20) or placebo (n = 10). Immune parameters were evaluated for differentiation of desensitized subjects from control subjects. RESULTS Subjects treated with dual SLIT had decreased rhinoconjunctivitis scores (P < .001) and medication use scores (P < .001) and reduced responses to TG and DM allergen based on results of skin prick tests or nasal disk challenges (P < .01 and P < .001, respectively) compared with placebo-treated control subjects. An increase in TG- and DM-specific IgG(4) levels, reduced allergen-specific IgE levels, and subsequent basophil activation were observed in the active treatment group. Dual SLIT promoted allergen-specific suppressive CD4(+)CD25(high)CD127(low)CD45RO(+) forkhead box protein 3 (Foxp3)(+) memory regulatory T cells with reduced DNA methylation of CpG sites within the Foxp3 locus. CONCLUSION The results of this pilot study suggest that dual SLIT could be an effective means to treat subjects with sensitivities to a variety of allergens and that long-term tolerance might be induced by epigenetic modifications of Foxp3 in memory regulatory T cells.