Accessory cells precondition naïve T cells and regulatory T cells for cytokine-mediated proliferation.

Naïve T cells and regulatory T cells, when purified, do not proliferate to the γc-cytokines IL-2, IL-7, or IL-15, despite their expression of cognate cytokine receptors. Dendritic cells (DCs) enabled the T cell proliferation to these cytokines, through cell-to-cell contact, but independent of T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in DC-depleted hosts. We propose calling this a "preconditioning effect". Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2 target genes. "Preconditioning" was necessary to activate these two pathways and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation, and prolonged S6 phosphorylation occurred. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.

The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models.

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × âˆ¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

Triple combination of BET plus PI3K and NF-κB inhibitors exhibit synergistic activity in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

Short-course IL-15 given as a continuous infusion led to a massive expansion of effective NK cells: implications for combination therapy with antitumor antibodies.

BACKGROUND: Full application of cytokines as oncoimmunotherapeutics requires identification of optimal regimens. Our initial effort with intravenous bolus recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions. Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10) provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8+ lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we shortened time of infusion. Treatment with rhIL-15 at a dose of 3-5 µg/kg as a 5-day continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while effector cell stimulation was comparable to the CIV-10 regimen. METHODS: Eleven patients with metastatic cancers were treated with rhIL-15 CIV-5, 3 µg (n=4), 4 µg (n=3), and 5 µg/kg/day (n=4) in a phase I dose-escalation study (April 6, 2012). RESULTS: Impressive expansions of NK cells were seen at all dose levels (mean 34-fold), including CD56bright NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8+ T cells (mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further testing. Cytolytic capacity of CD56bright and CD56dim NK cells was increased by interleukin-15 assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural killer group 2D-mediated cytotoxicity. The best response was stable disease. CONCLUSIONS: IL-15 administered as CIV-5 substantially expanded NK cells with increased cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their mechanism of action including alemtuzumab, obinutuzumab, avelumab, and mogamulizumab could benefit from those NK cell expansions and provide a promising therapeutic strategy. TRIAL REGISTRATION NUMBERS: NCT01572493, NCT03759184, NCT03905135, NCT04185220 and NCT02689453.

Enhanced efficacy of JAK1 inhibitor with mTORC1/C2 targeting in smoldering/chronic adult T cell leukemia.

Adult T-cell leukemia (ATL) is an aggressive T-cell lymphoproliferative malignancy of regulatory T lymphocytes (Tregs), caused by human T-cell lymphotropic virus 1 (HTLV-1). Interleukin 2 receptor alpha (IL-2Rα) is expressed in the leukemic cells of smoldering/chronic ATL patients, leading to constitutive activation of the JAK/STAT pathway and spontaneous proliferation. The PI3K/AKT/mTOR pathway also plays a critical role in ATL cell survival and proliferation. We previously performed a high-throughput screen that demonstrated additive/synergistic activity of Ruxolitinib, a JAK1/2 inhibitor, with AZD8055, an mTORC1/C2 inhibitor. However, effects of unintended JAK2 inhibition with Ruxolitinib limits it therapeutic potential for ATL patients, which lead us to evaluate a JAK1-specific inhibitor. Here, we demonstrated that Upadacitinib, a JAK-1 inhibitor, inhibited the proliferation of cytokine-dependent ATL cell lines and the expression of p-STAT5. Combinations of Upadacitinib with either AZD8055 or Sapanisertib, mTORC1/C2 inhibitors, showed anti-proliferative effects against cytokine-dependent ATL cell lines and synergistic effect with reducing tumor growth in NSG mice bearing IL-2 transgenic tumors. Importantly, the combination of these two agents inhibited ex vivo spontaneous proliferation of ATL cells from patients with smoldering/chronic ATL. Combined targeting of JAK/STAT and PI3K/AKT/mTOR pathways represents a promising therapeutic intervention for patients with smoldering/chronic ATL.

Electrochemical evidence for in situ microbial activity at the Deep Mine Microbial Observatory (DeMMO), South Dakota, USA.

The subsurface is Earth's largest reservoir of biomass. Micro-organisms are the dominant lifeforms in this habitat, but the nature of their in situ activities remains largely unresolved. At the Deep Mine Microbial Observatory (DeMMO) located in the Sanford Underground Research Facility (SURF) in Lead, South Dakota (USA), we performed in situ electrochemical incubations designed to assess the potential for deep groundwater microbial communities to utilize extracellular electron transfer to support microbial respiration. DeMMO 4 was chosen for its stable geochemistry and microbial community. Graphite and indium tin oxide electrodes poised at -200 mV versus SHE were incubated along with open circuit controls and various minerals in a parallel flow reactor that split access to fluids across different treatments. From the patterns of net current over time (fluctuating between anodic and cathodic currents over the course of a few days to weeks) and the catalytic features measured using periodic cyclic voltammetry, evidence of both oxidative and reductive microbe-electrode interactions was observed. The predominant catalytic activity ranged from -210 to -120 mV. The observed temporal variability in electrochemical activity was unexpected given the documented stability in major geochemical parameters. This suggests that the accessed fluids are more heterogeneous in electrochemically active microbial populations than previously predicted from the stable community composition. As previously reported, the fracture fluid and surface-attached microbial communities at SURF differed significantly. However, only minimal differences in community composition were observed between poised potential electrodes, open circuit electrodes, and mineral incubations. These data support that in this environment the ability to attach to surfaces is a stronger driver of microbial community structure than the type or reactivity of the surface. We demonstrate that insight into specific activities can be gained from electrochemical methods, specifically chronoamperometry coupled with routine cyclic voltammetry, which provide a sensitive approach to evaluate microbial activities in situ.

90Y-Daclizumab (Anti-CD25), High-Dose Carmustine, Etoposide, Cytarabine, and Melphalan Chemotherapy and Autologous Hematopoietic Stem Cell Transplant Yielded Sustained Complete Remissions in 4 Patients with Recurrent Hodgkin's Lymphoma.

Background: Despite advances in therapy of Hodgkin's lymphoma (HL), a proportion of patients will not respond or relapse. The authors had previously identified CD25, IL-2Rα, as a target for systemic radioimmunotherapy of HL since most normal cells do not express CD25, but it is expressed by a minority of Hodgkin/Reed-Sternberg (HRS) cells and most Tregs rosetting around HRS cells. Study Design and Treatment: This was a single institution, nonrandomized, open-label phase I/II trial of radiolabeled 90Y-daclizumab, an anti-CD25 monoclonal antibody, BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning treatment followed by autologous hematopoietic stem cell transplant (ASCT). Four patients with refractory and relapsed HL were treated in this trial with 3 patients receiving a single dose of 564.6-574.6 MBq 90Y-daclizumab and the fourth patient receiving two doses of 580.9-566.1 MBq 90Y-daclizumab followed by high-dose chemotherapy and ASCT. Results: All 4 evaluable patients treated with 90Y-daclizumab obtained complete responses (CRs) that are ongoing 4.5-7 years following their stem cell transplant. The spectrum and severity of adverse events were mild and more importantly none of the patients, including several with multiple therapies before this treatment, developed the myelodysplastic syndrome. Discussion: Targeting by daclizumab was not directed primarily at tumor cells, but rather the nonmalignant CD25-expressing T cells adjacent to the HRS cells and 90Y-daclizumab provided strong enough ß emissions to kill CD25-negative tumor cells at a distance by a crossfire effect. Furthermore, the strong ß irradiation killed normal cells in the tumor microenvironment. Conclusions: 90Y-daclizumab (anti-CD25), high-dose BEAM chemotherapy and ASCT was well tolerated and yielded sustained complete remissions in all 4 patients with recurrent HL patients who completed their treatment. Significance: Despite advances, a proportion of patients with HL will not have a CR to their initial treatment, and some with CRs will relapse. They demonstrated that the addition of 90Y-daclizumab into the preconditioning regimen for refractory and relapsed HL patients with high-dose BEAM chemotherapy and ASCT provided sustained CRs in the 4 patients studied. Two of these patients were highly refractory to multiple prior treatments with bulky disease at entry into this study, including 1 patient who never entered a remission and had failed 6 different therapeutic regimens. Despite the small number of patients treated in this study, the sustained clinical benefit in these patients indicates a highly effective treatment. The daclizumab was directed primarily not at HRS cells themselves but toward nonmalignant T cells rosetting around malignant cells. 90Y provided strong ß emissions that killed antigen nonexpressing tumor cells at a distance by a crossfire effect. Furthermore, the strong ß radiation killed normal cells in the tumor microenvironment that nurtured the malignant cells in the lymphomatous mass. The present study supports expanded analysis of 90Y-daclizumab as part of the regimen of ASCT in patients with refractory and relapsed HL.

Detecting Endogenous Microbial Metabolism and Differentiating Between Abiotic and Biotic Signals Observed by Bioelectrochemical Systems in Soils.

Unambiguous detection of chemical and physical signatures of microbial life on Mars or other solar system bodies requires differentiation between signals produced by biotic and abiotic processes; instruments aimed at generalized in situ extant life detection would therefore increase the science return of a life-detection mission. Here, we investigate Bioelectrochemical Systems (BES) as a technique to measure microbial metabolism (which produces electrical current and redox changes) and distinguish between potential abiotic and biotic responses in environmental samples. Samples from inhabited niches should contain everything necessary to produce current, that is, catalysts (microorganisms) and fuel (nutrients). BES can also probe for inactive organisms in less energetically rich areas by adding a fuel to drive metabolism. A commercial potting soil and a Mars simulant soil were inoculated in the anodic chamber of microbial fuel cells, and current was monitored over time. Addition of a fuel (electron donor) source was tested for metabolic stimulation of endogenous microbes. Redox reactions between Mars simulant soil and the introduced electron donor (lactate) produced false-positive results, emphasizing the importance of careful interpretation of signals obtained. The addition of lactate to both soils resulted in enhanced biologically produced current, allowing stimulation and detection of dormant microbes. Our results demonstrate that BES provide an approach to detect metabolism in samples without prior knowledge of the organisms present, and that thorough electrochemical analyses and experimental design are necessary to determine if signals are biotic.

Magnetic Resonance Elastography of the Liver in Children and Adolescents: Assessment of Regional Variations in Stiffness.

RATIONALE AND OBJECTIVES: We describe our experience in measuring parenchyma stiffness across the liver Couinaud segments in lieu of the conventional practice of using a single slice-wise "global" region-of-interest. We hypothesize that the heterogeneous nature of fibrosis can lead to regional stiffness within the organ, and that it can be reflected by Couinaud segment-based magnetic resonance elastography measurements. MATERIALS AND METHODS: This retrospective study involved from 173 patients (116 males, 57 females, 1.0-22.5 years, 14.7 ± 3.5 years) who underwent exams between June 2017 and September 2018. Liver stiffness across the eight Couinaud segments was measured in addition to a single-slice global measurement by two analysts. Inter- and intrarater analysis was performed in a subset of 20 cases. Individual segment stiffness values, the average across the segments, and the coefficients of variation (CoV) were compared to global single-slice-derived values using linear and Lin's concordance correlation coefficients. Linear correlations between stiffness values versus age, gender, and body-mass-index (BMI) were also evaluated. RESULTS: We observed CoVs ranging from 3.1%-79.2%, 17.2 ± 7.2%. The CoV was not correlated with age or BMI (r2 < 0.01, p = 0.99 for both). The CoV did not differ between males (17.1 ± 5.6%) and females (17.3 ± 9.8%) (p = 0.88). There were no correlations between global stiffness versus age (r2 = 0.02, p = 0.84) or BMI (r2 = 0.03, p = 0.68). A range of 0.58-0.86 was observed for Lin's concordance correlation coefficient between segmental stiffness, the average stiffness across segments, and global stiffness. Segments II and VII had the highest frequency of being the stiffest Couinaud segment. The average stiffness across the segments correlated strongly with the single-slice global measurement (r2 = 0.88, p< 0.01). CONCLUSION: There exists potential variations in parenchyma stiffness across the liver Couinaud segments, which may reflect the heterogeneous nature of fibrosis. This variation can potentially provide additional diagnostic and clinical information.

Differences in Applied Redox Potential on Cathodes Enrich for Diverse Electrochemically Active Microbial Isolates From a Marine Sediment.

The diversity of microbially mediated redox processes that occur in marine sediments is likely underestimated, especially with respect to the metabolisms that involve solid substrate electron donors or acceptors. Though electrochemical studies that utilize poised potential electrodes as a surrogate for solid substrate or mineral interactions have shed some much needed light on these areas, these studies have traditionally been limited to one redox potential or metabolic condition. This work seeks to uncover the diversity of microbes capable of accepting cathodic electrons from a marine sediment utilizing a range of redox potentials, by coupling electrochemical enrichment approaches to microbial cultivation and isolation techniques. Five lab-scale three-electrode electrochemical systems were constructed, using electrodes that were initially incubated in marine sediment at cathodic or electron-donating voltages (five redox potentials between -400 and -750 mV versus Ag/AgCl) as energy sources for enrichment. Electron uptake was monitored in the laboratory bioreactors and linked to the reduction of supplied terminal electron acceptors (nitrate or sulfate). Enriched communities exhibited differences in community structure dependent on poised redox potential and terminal electron acceptor used. Further cultivation of microbes was conducted using media with reduced iron (Fe0, FeCl2) and sulfur (S0) compounds as electron donors, resulting in the isolation of six electrochemically active strains. The isolates belong to the genera Vallitalea of the Clostridia, Arcobacter of the Epsilonproteobacteria, Desulfovibrio of the Deltaproteobacteria, and Vibrio and Marinobacter of the Gammaproteobacteria. Electrochemical characterization of the isolates with cyclic voltammetry yielded a wide range of midpoint potentials (99.20 to -389.1 mV versus Ag/AgCl), indicating diverse metabolic pathways likely support the observed electron uptake. Our work demonstrates culturing under various electrochemical and geochemical regimes allows for enhanced cultivation of diverse cathode-oxidizing microbes from one environmental system. Understanding the mechanisms of solid substrate oxidation from environmental microbes will further elucidation of the ecological relevance of these electron transfer interactions with implications for microbe-electrode technologies.

Clinical trial of a humanized anti-IL-2/IL-15 receptor ß chain in HAM/TSP.

OBJECTIVE: Human T cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neurological disease. Chronic activation of CD8+ T cells, as evidenced by increased spontaneous lymphoproliferation and HTLV-1-specific cytotoxic T cells, has been demonstrated in HAM/TSP patients. Since IL-2 and IL-15 stimulate memory CD8+ T cell activity, these cytokines have been implicated in the immunopathogenesis of HAM/TSP. In this phase I trial, we evaluated the safety, pharmacokinetics, and ability of Hu-Mikß1, a humanized monoclonal antibody directed toward the IL-2/IL-15 receptor ß-chain (IL-2/IL-15Rß: CD122), to saturate CD122 and regulate abnormal immune responses in patients with HAM/TSP by inhibition of IL-15 action. METHODS: Hu-Mikß1 was administered intravenously at doses of 0.5 mg/kg, 1.0 mg/kg, or 1.5 mg/kg in a total of nine HAM/TSP patients. Five doses of Hu-Mikß1 were administered at 3-week intervals. The clinical response was evaluated using standardized scales. Viral and immunologic outcome measures were examined including HTLV-1 proviral load, T cell phenotypic analysis and spontaneous lymphoproliferation in HAM/TSP patients. RESULTS: There was no significant toxicity associated with Hu-Mikß1 administration in HAM/TSP patients. Saturation of CD122 by Hu-Mikß1 was achieved in five out of nine HAM/TSP patients. Administration of Hu-Mikß1 was associated with inhibition of aberrant CD8+ T cell function including spontaneous lymphoproliferation and degranulation and IFN-γ expression, especially in HAM/TSP patients that achieved CD122 saturation. INTERPRETATION: The treatment with Hu-Mikß1 had a number of immunological effects on HAM/TSP patients although no clinical efficacy was observed. We also did not see any dose-related toxicity.

IL15 by Continuous Intravenous Infusion to Adult Patients with Solid Tumors in a Phase I Trial Induced Dramatic NK-Cell Subset Expansion.

PURPOSE: The first-in-human clinical trial with human bolus intravenous infusion IL15 (rhIL15) was limited by treatment-associated toxicity. Here, we report toxicity, immunomodulation, and clinical activity of rhIL15 administered as a 10-day continuous intravenous infusion (CIV) to patients with cancers in a phase I trial. PATIENTS AND METHODS: Patients received treatment for 10 days with CIV rhIL15 in doses of 0.125, 0.25, 0.5, 1, 2, or 4 µg/kg/day. Correlative laboratory tests included IL15 pharmacokinetic (PK) analyses, and assessment of changes in lymphocyte subset numbers. RESULTS: Twenty-seven patients were treated with rhIL15; 2 µg/kg/day was identified as the MTD. There were eight serious adverse events including two bleeding events, papilledema, uveitis, pneumonitis, duodenal erosions, and two deaths (one due to likely drug-related gastrointestinal ischemia). Evidence of antitumor effects was observed in several patients, but stable disease was the best response noted. Patients in the 2 µg/kg/day group had a 5.8-fold increase in number of circulating CD8+ T cells, 38-fold increase in total NK cells, and 358-fold increase in CD56bright NK cells. Serum IL15 concentrations were markedly lower during the last 3 days of infusion. CONCLUSIONS: This phase I trial identified the MTD for CIV rhIL15 and defined a treatment regimen that produced significant expansions of CD8+ T and NK effector cells in circulation and tumor deposits. This regimen has identified several biological features, including dramatic increases in numbers of NK cells, supporting trials of IL15 with anticancer mAbs to increase antibody-dependent cell-mediated cytotoxicity and anticancer efficacy.

Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T Cell Leukemia/Lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.

Variation in electrode redox potential selects for different microorganisms under cathodic current flow from electrodes in marine sediments.

Extracellular electron transport (EET) is a microbial process that allows microorganisms to transport electrons to and from insoluble substrates outside of the cell. Although progress has been made in understanding how microbes transfer electrons to insoluble substrates, the process of receiving electrons has largely remained unexplored. We investigated redox potentials favourable for donating electrons to dissolved and insoluble components in Catalina Harbor marine sediment by combining electrochemical techniques with geochemistry and molecular methods. Working electrodes buried in sediment microcosms were poised at seven redox potentials between -300 and -750 mV versus Ag/AgCl using a three-electrode system. In electrode biofilms recovered after 2-month incubations, overall community diversity increased with more negative redox potentials. Abundances of known EET-capable groups (e.g., Alteromonadales and Desulfuromonadales) varied with redox potential. Motility and chemotaxis genes were found in greater abundance in electrode communities, suggesting a possible selective advantage of these pathways for colonization and utilization of the electrode. Our enrichments demonstrated the validity of this approach in capturing groups known, as well as novel groups (e.g., Campylobacterales) that perform EET. The diverse nature of the enriched cathode communities suggest that insoluble substrate oxidation may be a critical, although poorly described microbial metabolic process in marine sediment.

IL15 Infusion of Cancer Patients Expands the Subpopulation of Cytotoxic CD56bright NK Cells and Increases NK-Cell Cytokine Release Capabilities.

The cytokine IL15 is required for survival and activation of natural killer (NK) cells as well as expansion of NK-cell populations. Here, we compare the effects of continuous IL15 infusions on NK-cell subpopulations in cancer patients. Infusions affected the CD56bright NK-cell subpopulation in that the expansion rates exceeded those of CD56dim NK-cell populations with a 350-fold increase in their total cell numbers compared with 20-fold expansion for the CD56dim subset. CD56bright NK cells responded with increased cytokine release to various stimuli, as expected given their immunoregulatory functions. Moreover, CD56bright NK cells gained the ability to kill various target cells at levels that are typical for CD56dim NK cells. Some increased cytotoxic activities were also observed for CD56dim NK cells. IL15 infusions induced expression changes on the surface of both NK-cell subsets, resulting in a previously undescribed and similar phenotype. These data suggest that IL15 infusions expand and arm CD56bright NK cells that alone or in combination with tumor-targeting antibodies may be useful in the treatment of cancer. Cancer Immunol Res; 5(10); 929-38. ©2017 AACR.

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

90Y-daclizumab, an anti-CD25 monoclonal antibody, provided responses in 50% of patients with relapsed Hodgkin's lymphoma.

Despite significant advances in the treatment of Hodgkin's lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed-Sternberg cells and by most polyclonal T cells rosetting around Reed-Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody (90)Y-daclizumab. (90)Y provides strong ß emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with (90)Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed-Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25(-) provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated (90)Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed-Sternberg cells provided meaningful therapy for select HL patients.

Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer.

PURPOSE: Interleukin-15 (IL-15) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-15 (rhIL-15) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy. PATIENTS AND METHODS: We performed a first in-human trial of Escherichia coli-produced rhIL-15. Bolus infusions of 3.0, 1.0, and 0.3 µg/kg per day of IL-15 were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer. RESULTS: Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 µg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 µg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. CONCLUSION: IL-15 could be safely administered to patients with metastatic malignancy. IL-15 administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-15 administration.

Phase 1 trial of IL-15 trans presentation blockade using humanized Mikß1 mAb in patients with T-cell large granular lymphocytic leukemia.

In the present study, Hu-Mikß1, a humanized mAb directed at the shared IL-2/IL-15Rß subunit (CD122) was evaluated in patients with T-cell large granular lymphocytic (T-LGL) leukemia. Hu-Mikß1 blocked the trans presentation of IL-15 to T cells expressing IL-2/IL-15Rß and the common γ-chain (CD132), but did not block IL-15 action in cells that expressed the heterotrimeric IL-15 receptor in cis. There was no significant toxicity associated with Hu-Mikß1 administration in patients with T-LGL leukemia, but no major clinical responses were observed. One patient who had previously received murine Mikß1 developed a measurable Ab response to the infused Ab. Nevertheless, the safety profile of this first in-human study of the humanized mAb to IL-2/IL-15Rß (CD122) supports its evaluation in disorders such as refractory celiac disease, in which IL-15 and its receptor have been proposed to play a critical role in the pathogenesis and maintenance of disease activity.

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.