Double Negative Research Articles

PO-393 Notch3 and CXCR4 cross-signalling sustains acute T-cell leukaemia progression

IntroductionAcute T-cell lymphoblastic leukaemia (T-ALL) is a childhood cancer, characterised by infiltration of immature T-cells in bone marrow. Notch hyperactivation is a major driver of T-ALL development where CXCL12/CXCR4 axis plays an important role in T-ALL maintenance. In thymus the lympho-stromal communication drives progressive maturation of T-cells. Notch receptors regulate T-cell fate choices, dominating early steps of thymocyte maturation. In T-cell differentiation, Notch3, in association with pre-TCR and chemochine receptor CXCR4, govern the transition from double negative (DN) to double positive (DP) thymocytes. Previously, our laboratory demonstrated the lymphomagenic potential of Notch3 by creating a transgenic mouse model (N3-ICtg), characterised by the constitutive activation of the intracellular domain (IC) of Notch3 receptor (N3-IC) in immature thymocytes. In order to investigate the oncogenic cross-talk between Notch3 and CXCR4 in T-ALL progression, we analysed DP T-cells in different lymphoid compartments of N3-ICtg mice.Material and methodsFreshly isolated cells from thymus, blood and bone marrow of N3-ICtg and WT mice were analysed by flow cytometry in order to verify the presence of DP T-cells and their cell-surface expression of CXCR4 and Notch3 receptors. Experiments in TALL1, a human T-ALL leukemic CD3+/CD4+/CD8+ cell line characterised by the activation of Notch3 and high expression of CXCR4, were also performed. TALL1 cells were treated with γ-secretase inhibitor (GSI) or their gene expression of Notch3 was silenced and then analysed by flow cytometry, RT-PCR and western blot. Statistical interpretation of the results was performed.Results and discussionsDP-gated thymocytes obtained by N3-ICtg mice have shown a high co-expression of Notch3 and CXCR4 and a high migratory ability induced by SDF-1. An anomalous percentage representation of these DP T-cells at different ages in circulating blood, spleen and bone morrow may suggest an interaction between CXCR4 and Notch3 in T-ALL cell propagation. Experiments in human TALL1 cell line with Notch3 targeted inhibition suggest a modulated expression of CXCR4 through a β-arrestin1-mediated mechanism. CXCR4-antagonists treatment will further elucidate the molecular crosstalk between the two receptors.ConclusionNotch3 abnormal pathway, through boosting the expression of CXCR4 on cell-surface, may play a role in DP T-cells egress from thymus, and define a possible mechanism of ’pre-leukemic-cells’ dissemination.

Abstract 294: BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL

Abstract BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T and B cells we examined peripheral blood counts in mice with T or B cell-specific deletion of Bim. In contrast to CD19CRE Bimfl/fl animals, LCKCRE Bimfl/fl mice had pronounced early lymphocytosis followed by lymphocyte count normalization as they aged. To explore BCL-2 family specific mechanism(s) leading to this normalization we compared cell death sensitivity and BCL-2 expression patterns of young versus old LCKCRE Bimfl/fl T cells. Young (6-12 weeks) and old (>40 weeks) LCKCRE Bimfl/fl mice had consistently abnormal thymocyte development hallmarked by a decrease in CD4+8+ double positive (DP) cells and accumulation of double negative (DN) and single positive (SP) cells as previously described in global Bim knockout mice. Thymocytes maintained cell death resistance to a number of apoptotic stimuli throughout all ages, reflecting a consistent loss of BIM. In contrast to thymocytes, mature splenic-derived LCKCRE Bimfl/fl T cells from young mice were equally sensitive to apoptotic stimuli as WT controls, indicating BCL-2 family compensation. Expression profiling in these cells revealed upregulation of other BH3-only proteins, including Noxa = Puma << Bmf, suggesting that these proteins functionally compensated for the loss of BIM. To determine if this was a T cell-intrinsic process and if the presence of other BH3-only proteins with pan-BCL-2 family binding affinities were required for lymphocyte number and apoptotic normalization, we analyzed peripheral blood from mice globally deleted in Bim, Bid, and Puma (TKO). TKO, like LCKCRE Bimfl/fl mice, developed profound early lymphocytosis followed by normalization as they aged. A subset of TKO mice developed T-ALL hallmarked by upregulation of Bmf and Bcl-w, an under characterized anti-apoptotic BCL-2 family protein, suggesting that BCL-W allowed for malignant T cell survival despite the presence of BMF. To determine if such a relationship exists in human disease, expression profiling of pediatric T-ALL patient samples revealed a significant negative correlation between BIM and BCL-W expression in developmentally immature and mature T-ALL. Our findings suggest a model in which the loss of BIM in T cells can be compensated for through upregulation of BMF, a BH3-only protein mechanistically similar to BIM. Loss of other ‘direct activating' BH3-only proteins increases the chances of malignant cell escape despite BMF upregulation, particularly when BCL-W is overexpressed. This work provides a developmental framework between BIM and BMF in T cells and for a BIM:BCL-W axis in T-ALL. Overexpression of BCL-W in BIMlow-expressing T-ALL may be an additional target responsible for early immature T-ALL therapeutic resistance. Citation Format: Lindsey M. Ludwig, Lauren E. Roach, Samuel G. Katz, Jill K. Fisher, Melissa Burns, Matthew R. Schnorenberg, Riyue Bao, Makda Zewde, Yusuke Nakamura, Alejandro Gutierrez, Loren D. Walensky, James L. LaBelle. BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 294.

A natural killer T-cell subset that protects against airway hyperreactivity

Infection of suckling mice with influenza virus expands a CD4-CD8- double-negative (DN) natural killer T(NKT) cell subpopulation that protects the mice as adults against allergen-induced airway hyperreactivity (AHR). However, this NKT cell subset has not been characterized, and the underlying mechanisms of protection remain unknown. We characterized this specific NKT cell subpopulation that developed during influenza infection in neonatal mice and that suppressed the subsequent development of AHR. A cell-surface marker was identified by comparing the mRNA expression profile of wild-type CD4+ NKT cells with that of suppressive Vα14 DN NKT cells. The marker-enriched NKT cell subset was then analyzed for its cytokine profile and its suppressive invitro and invivo abilities. We showed that DN NKT cells with high CD38 expression produced IFN-γ, but not IL-17, IL-4, or IL-13, and inhibited development of AHR through contact-dependent suppression of helper CD4 T-cell proliferation. The NKT subset expanded in the lungs of neonatal mice after infection with influenza and also after treatment of neonatal mice with Nu-α-GalCer, which effectively increased DN CD38hi NKT cell numbers. These results suggest that early/neonatal exposure to infection or antigen challenge affects subsequent lung immunity by altering the cellular composition of cells in the lung and that some subsets of NKT cells suppress AHR. These results provide a possible mechanism by which prior infections can protect against the development of allergic asthma and might be further explored as a protective measure for young children.

A BTB-ZF protein, ZNF131, is required for early B cell development

Members of the BTB-ZF transcription factor family play important roles in lymphocyte development. During T cell development, ZNF131, a BTB-ZF protein, is critical for the double-negative (DN) to double-positive (DP) transition and is also involved in cell proliferation. Here, we report that knockout of Znf131 at the pre-pro-B cell stage in mb1-Cre knock-in mouse resulted in defect of pro-B to pre-B cell transition. ZNF131 was shown to be required for efficient pro-B cell proliferation as well as for immunoglobulin heavy chain gene rearrangement that occurs in the proliferating pro-B cells. We speculate that inefficient gene rearrangement may be due to loss of cell proliferation, since cell cycle progression and immunoglobulin gene rearrangement, which would occur in a mutually exclusive manner, may be interconnected or coupled to avoid occurrence of genomic instability. ZNF131 suppresses expression of Cdk inhibitor, p21cip1, and that of pro-apoptotic factors, Bax and Puma, targets of p53, to facilitate cell cycle progression and suppress unnecessary apoptosis, respectively, of pro-B cells. There results demonstrate the essential roles of ZNF131 in coordinating the B cell differentiation and proliferation.

Thymic selection of innate self-specific T cells: Both Timing and Signal Strength matter

Abstract Naïve CD4 and CD8 T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals are crucial for installing a quiescent program that guides them to the lymph nodes yet allows for immune activation upon encounter of high-affinity ligands in the periphery. In contrast, agonist-selected T cells receive strong self-based TCR signals in the thymus that imprint them to guard the mucosal borders and maintain quiescence at steady state. Although the TCR signal strength is a decisive factor in instructing these different outcomes, the mechanism that generates protective self-specific T cells remains largely enigmatic. Previous work suggests that the self-specific double negative (DN) T cell lineage already diverges early during T cell development. We have explored the role of developmental timing by Cre-mediated deletion of components of the TCR signaling pathway. The Lck proximal promoter drives Cre expression before b-selection and the CD4 promoter drives Cre expression after b-selection but before positive selection. As TCR targets we investigated the lack of Themis expression, essential for the generation of conventional naïve T cells, but dispensable for agonist selection. Vice versa, we analyzed the requirement for Stim1 and Stim2, driving agonist selection, but not conventional selection. Both Lck prox Cre- and CD4 Cre-driven deletion of Themis lead to a drastic decrease in naïve T cells. However, DN T cells are particularly dependent on early strong TCR signals at the Pre-TCR stage, as only Lck prox Cre- but not CD4 Cre-driven deletion of Stim1/2 eliminates this subset. Our results suggest that strong signals at the Pre-TCR checkpoint are crucial for the generation of innate self-specific T cells.

Stage specific requirement of TCF-1 for restraining Th17 immunity

Abstract Besides the positive and negative selection for shaping T cell repertoire that only for attacking foreign antigens, central immune system also control the type and scale of immune responses in the periphery. T cell factor 1 (TCF-1) is highly expressed in the early thymic progenitors and has been shown to restrain Th17 responses in the periphery. However, it is not well understood which specific T cell developmental stage is critical for TCF-1 to determine the magnitude of Th17 immunity in the periphery. Here we showed that conditional deletion of TCF-1 early at hematopoietic, but not CD4+CD8+ double positive (DP) stage, resulted in severe reduction of thymic cellularity, enhanced Th17 differentiation and more aggravated experimental autoimmune encephalomyelitis (EAE). We also observed abnormal IL-17 expression in developing CD4−CD8− double negative (DN) thymocytes. Expression of wild-type TCF-1 in CD4−CD8− DN thymocytes, but not peripheral mature T cells, inhibited IL-17 expression. We also demonstrated that TCF-1 binds to IL-17 promoter region to perform its histone deacetylase (HDAC) function and hence restrain IL-17 expression in thymus. Our study first demonstrated the stage specific requirement of TCF-1 to restrain peripheral Th17 responses and thus provide an additional way to develop treatment for autoimmune diseases.

A requirement for Lysine-specific histone demethylase 1A (Lsd1) for T cell development

Abstract Lysine-specific demethylase 1A (Lsd1), also known as Kdm1a, is a part of the histone demethylase family of enzymes. Histone demethylases are key epigenetic regulatory proteins of the mammalian genome that participate in chromatin remodeling. It has been shown that Lsd1 mediates H3K4me2/me1 histone demethylation, and this event has been connected with gene repression in hematopoietic stem cells (HSC) and HSC maintenance. While Lsd1’s role in plasma cell differentiation and as a therapeutic target in cancer are well established, its role in T cell development has not been examined. To investigate Lsd1’s role in T cell development, we used the CD2i-Cre system to conditionally delete Lsd1 in immature double negative (DN) thymocytes. Here we show that deletion of Lsd1 causes a severe reduction in total thymocyte numbers attributable to a defect at the CD8+CD4−CD3lo intermediate single positive (ISP) stage. In addition, Lsd1 deficient T cells are reduced in number, exhibit a memory phenotype, and express low surface levels of T cell antigen receptor (TCR) as assessed by TCRβ and CD3 staining. Together, these findings identify an important role for Lsd1 in T cell maturation and indicate that regulation of H3K4 histone demethylation is essential at several critical stages of thymocyte development.

Genetic interaction between two insulin-dependent diabetes susceptibility loci, Idd2 and Idd13, in determining immunoregulatory DN T cell proportion.

Several immune regulatory cell types participate in the protection against autoimmune diseases such as autoimmune diabetes. Of these immunoregulatory cells, we and others have shown that peripheral CD4-CD8- double negative (DN) T cells can induce antigen-specific immune tolerance. Particularly, we have described that diabetes-prone mice exhibit a lower number of peripheral DN T cells compared to diabetes-resistant mice. Identifying the molecular pathways that influence the size of the DN T cell pool in peripheral lymphoid organs may thus be of interest for maintaining antigen-specific immune tolerance. Hence, through immunogenetic approaches, we found that two genetic loci linked to autoimmune diabetes susceptibility, namely Idd2 and Idd13, independently contribute to the partial restoration of DN T cell proportion in secondary lymphoid organs. We now extend these findings to show an interaction between the Idd2 and Idd13 loci in determining the number of DN T cells in secondary lymphoid organs. Using bioinformatics tools, we link potential biological pathways arising from interactions of genes encoded within the two loci. By focusing on cell cycle, we validate that both the Idd2 and Idd13 loci influence RAD51 expression as well as DN T cell progression through the cell cycle. Altogether, we find that genetic interactions between Idd2 and Idd13 loci modulate cell cycle progression, which contributes, at least in part, to defining the proportion of DN T cells in secondary lymphoid organs.

Global dynamics of stage-specific transcription factor binding during thymocyte development

In vertebrates, multiple transcription factors (TFs) bind to gene regulatory elements (promoters, enhancers, and silencers) to execute developmental expression changes. ChIP experiments are often used to identify where TFs bind to regulatory elements in the genome, but the requirement of TF-specific antibodies hampers analyses of tens of TFs at multiple loci. Here we tested whether TF binding predictions using ATAC-seq can be used to infer the identity of TFs that bind to functionally validated enhancers of the Cd4, Cd8, and Gata3 genes in thymocytes. We performed ATAC-seq at four distinct stages of development in mouse thymus, probing the chromatin accessibility landscape in double negative (DN), double positive (DP), CD4 single positive (SP4) and CD8 SP (SP8) thymocytes. Integration of chromatin accessibility with TF motifs genome-wide allowed us to infer stage-specific occupied TF binding sites within known and potentially novel regulatory elements. Our results provide genome-wide stage-specific T cell open chromatin profiles, and allow the identification of candidate TFs that drive thymocyte differentiation at each developmental stage.

Genetic Deletion of P2Y2 Receptor Suppresses Lithium‐induced Medullary Collecting Duct Remodeling in Mice

Previously we reported that genetic deletion of P2Y2 receptor (R) significantly protects against lithium (Li)-induced nephrogenic diabetes insipidus (NDI) by suppressing polyuria, natriuresis and kaliuresis even on long-term basis (5 months). Li administration also results in collecting duct remodeling, whereby the proportion of H+-ATPase-positive intercalated cells (IC) increase relative to the proportion of AQP2-positive principal cells (PC). Hence, we investigated whether P2Y2-R deletion has a significant effect on Li-induced medullary collecting duct (mCD) remodeling. Li was administered to wild type (WT) and P2Y2-R knockout (KO) mice (both in B6D2 genetic background) embedded in food for either 5 months (40 mmol LiCl/kg chow) or 30 days (40 mmol/kg chow for 7 days followed by 60 mmol LiCl/kg chow), and humanely euthanized. Using confocal immunofluorescence microscopy and double labeling, kidney sections from 4 to 7 mice per group were examined for the percent of cells labeled for AQP2 or H+-ATPase or none. Cell proliferation was assessed by immunolabeling for Ki67. Bar graph below shows the results for collecting duct remodeling. In both series, Li feeding significantly (P < 0.05) decreased the percent of PC and increased IC cells in the WT but not in the KO mice. In both series, Li-treatment resulted in significant (P < 0.05) differences in the PC and IC proportions between WT and KO mice. Furthermore, Li-induced differences in PC/IC ratio in WT mice were more marked in the long-term series. In the high-dose study Li-induced higher percent of double negative (DN) cells in the KO mice, but not in the WT mice. Li treatment caused significant increase in Ki67 labeling in PC and IC cells in WT, but not in KO mice, indicating that proliferation of both cell types contribute to CD remodeling. These results suggest that P2Y2-R plays a significant role in Li-induced conversion of PC to IC cells, and thus shed new light on the cell biology of collecting duct remodeling. Support or Funding Information Department of Veterans Affairs; National Institutes of Health This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic Ablation of the ABC Transporter Abcc4 Impairs Lymphoid Leukemogenesis

ABCC4 is an ABC transporter of the C sub‐family that is ubiquitously expressed and exports key endogenous compounds including cAMP and prostaglandins, and cancer chemotherapeutic drugs. cAMP and prostaglandins are signaling molecules that, among other functions, regulate thymocyte development and function. Thymocyte progenitors, IL7 receptor positive cells, migrate from the bone marrow to the thymus and give rise to mature T‐cells. The maturation of thymocytes can be followed by monitoring for the presence of surface markers CD4 and CD8. Briefly, thymocytes are initially double negative (DN) cells, with neither CD4 nor CD8; after T‐cell receptor rearrangement, CD4 and CD8 are expressed on double positive cells (DP), which following positive and negative selection, become mature, single positive (SP) cells expressing either CD4 or CD8. Abcc4 is highly expressed in DP and CD4‐positve thymocytes, while being undetectable in DN thymocytes from wild‐type (WT) mice, suggesting a developmental regulation of Abcc4. Therefore, we hypothesized that ablation of Abcc4 from mice would lead to aberrant T‐cell development and function.ABCC4 absence had no measureable impact on the number of hematopoietic progenitors. We hypothesized that lymphoid progenitors in Abcc4‐knockout(KO) mice may have an impaired leukemogenesis potential. To test this hypothesis, hematopoietic progenitors from Abcc4‐KO or WT mice were transduced with a retrovirus harboring a MYCN cDNA, a known driver of lymphoid leukemia, and subsequently transplanted into lethally irradiated congenic WT recipient mice. Mice transplanted with either WT or Abcc4‐KO MYCN‐transduced hematopoietic progenitors developed both B‐ and T‐cell leukemia, with tumors expressing Abcc4 on the plasma membrane. Kaplan‐Meier survival analysis revealed that mice transduced with Abcc4‐KO progenitors had a dramatic increase in median survival from 81.5 days in mice who received WT progenitors to 112 days in mice who received Abcc4‐KO progenitors. These studies suggested the lymphoid progenitors from Abcc4‐KO mice harbored an intrinsic defect manifested by reduced lymphoid leukemia. This was further investigated by performing a competitive bone‐marrow transplantation experiment. Briefly, hematopoietic progenitors from Abcc4‐KO and wildtype mice were isolated and mixed in equal numbers (each harboring a different surface marker to enable identification), then transplanted into congenic wildtype mice following lethal irradiation; subsequently the differentiated lymphoid progeny were identified in the peripheral blood. These studies revealed that the number of CD4+ cells from Abcc4‐KO donors was reduced, thus confirming a defect in Abcc4‐KO lymphoid progenitors. A defect in peripheral thymocyte function is also a potential mechanism, as genes in the arachidonic acid pathway regulate further differentiation and function of mature thymocytes. We discovered altered expression of genes in the arachidonic acid pathway such as Cox‐1 and Ptger4, when compared to age‐matched WT mice. These data suggest that genetic ablation of Abcc4 has an effect on arachidonic acid signaling, which may in turn affect thymocyte function.Overall, the data presented herein suggest that ABCC4 impacts lymphoid progenitors and differentiated thymocytes. One implication of these findings is that deficiency or insufficiency of ABCC4 may reduce the possibility of developing lymphoid malignancies.Support or Funding InformationThis work was funded by NIH and ALSACThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

BackgroundSystemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA+ SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6+Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity.MethodsWe recruited 25 anti-DNA+ and 25 anti-DNA− treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6+ Th cells and their additional subsets were analyzed in each patient by flow cytometry.ResultsAnti-DNA+ SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6+ Th cell subsets showed that anti-DNA+ SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6− Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6+ Th subsets and clinical indicators, specifically in anti-DNA+ SLE patients.ConclusionsOur data indicated that CCR6+ Th cells and their subsets were elevated and correlated with disease activity in anti-DNA+ SLE patients. We speculated that CCR6+ Th cells may contribute to distinct disease severity in anti-DNA+ SLE patients.

Modified H-shaped DNG metamaterial for multiband microwave application

A modified H-shaped double negative (DNG) metamaterial unit cell aimed at X- and Ku-band applications has been presented. Combination of rectangular split ring resonator and complementary H-shaped resonator has been used to construct the unit cell. A multiband DNG bandwidth of 0.845 GHz (7.615–8.46 GHz), 0.605 GHz in X band (8.755–9.36 GHz), and 4.32 GHz in Ku-band (10.68–15 GHz) has been achieved by the unit cell. Moreover, the unit cell has realized a compact size of 0.792 cm2, which is the smallest reported size with the DNG bandwidth more than 4 GHz in a particular band. Commercially available finite difference time domain (FDTD)-based computer simulation technology (CST) has been used for designing the unit cell. Effect of different parts of the unit cell on resonance characteristics has also been alluded. Different array structures have been designed and analyzed for the verification of DNG characteristics. 4 × 4 array has been fabricated which shows close likelihood with the simulated results. Simulated and measured results confirm the effectual involvement in X- and Ku-band applications.

Dielectric Materials Containing Active Dielectric-Metal Composite Nanoparticles as Double Negative Materials in the Visible

In the visible spectral range, Mie resonance-based double negativity has still not been observed in all-dielectric metamaterials due to the lack of dielectrics with sufficiently high refractive indices. In this work, the dielectric materials containing nanospheres of gain-metal nanocomposites are proposed as double negative materials operating in the visible. They are embedded with two types of composite nanospheres: one contributes to the negative permeability via magnetic Mie resonance facilitated by gain-assisted high indices of composite nanospheres and the other one to the negative permittivity via plasmonic dipolar resonance in the same spectral range. The figure-of-merit of these materials reaches up to infinity or even can be negative, implying the gain, depending on the structural and material parameters. The proposed materials can find broad applications as loss-free negative index materials in the visible and near-infrared.

Abstract TP253: Double Negative T Cells With FasL Mutation Promote M2-Microglial Polarization By Inhibiting PTPN2/TNF-α Pathway in Experimental Stroke

Instructions: The crosstalk between T lymphocytes and microglia plays an important role in modulating neuroinflammation after ischemic stroke. We have demonstrated FasL mutant ( gld ) mice attenuated brain inflammation after ischemic injury, with a dramatic accumulation of double negative (DN) T cells in brain. However, the effects of DN T lymphocytes in modulating pro-inflammatory (M1) or anti-inflammatory (M2) microglial polarization are not fully understood. Methods: Middle cerebral artery occlusion (MCAO) was induced for 60 min followed by 24 h and 72 h reperfusion in both gld and C57BL/6J mice. DN T lymphocytes isolated from gld and C57BL/6J mice after MCAO were cocultured with primary microglia for 24 h. The subtypes of T lymphocytes and microglial polarizations in ischemic brain were measured by flow cytometry, real-time PCR and immunofluorescence.Oxygen glucose deprivation (OGD)-induced neurons were treated by the coculture supernatant for 24 h, followed by propidium iodide and calcein AM staining. In inhibition study, DN T cells were pretreated with ethyl-3,4-dephostatin, a inhibitor of protein tyrosine phosphatases nonreceptor type 2 (PTPN2), for 24 h. Results: In vivo , compared to C57BL/6J mice, DN T cells were significantly accumulated in ischemic cerebral hemisphere in gld mice 72 h after MCAO, accompanied by increased M2 microglial polarization. In vitro , microglia underwent M1 polarization when cocultured with DN T cells from C57BL/6J mice, whereas shifted to M2 phenotype when cocultured with gld -derived DN T cells. In addition, the coculture supernatant from gld mice attenuated OGD-induced neuronal death and neurotoxicity. Furthermore, DN T lymphocytes from gld mice exhibited a profound downregulation of PTPN2 in protein levels, which was accompanied by an enhanced protein level of TNF-α. In contrast, inhibition of PTPN2 abolished the decreased expression of TNF-α in DN T cells from gld mice, and simultaneously reduced the M2-microlgial polarization. Conclusions: DN T cells with FasL mutation drives M2-microglial polarization and attenuated neuroinflammation in experimental stroke. The underlying mechanisms may involve the abolishment of FasL/PTPN2 pathway in inhibiting TNF-α expression in DN T cells with FasL mutation.

AIF loss deregulates hematopoiesis and reveals different adaptive metabolic responses in bone marrow cells and thymocytes.

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid β-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid β-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.

Microstrip Sierpinski fractal carpet for slot antenna with metamaterial loads for dual-band wireless application

The compact microstrip antennas with the dual-band characteristic and reconfigurable qualification have been noticed and investigated in this paper. We have developed the slot microstrip antenna with Sierpinski carpet and Minkowski formations and then the metamaterial loads are placed on this antenna in the slot area. The reflection/transmission method has considered to obtain the permittivity and permeability of the split ring resonator (SRR) as a metamaterial. Finally, we show that by using some junction in the metamaterial layer, we can obtain reconfigurable characteristic. Both antennas have dual-band characteristic where the first antenna has two resonances at 3.2 and 4.5 GHz and in the second antenna by altering the metamaterial layer both resonances are occurring at 3.5 and 5.8 GHz. We show that the antenna has two resonance frequencies that matched with the metamaterial Double Negative (DNG) characteristic. Both antennas are printed on FR-4 also we compared measurement and simulation results together. Aforementioned antennas cover the main wireless, WiMAX and LTE bands with sufficient gain and high efficiency (more than 80% typically) with a bi-directional radiation pattern for the indoor application. Here, the metamaterial is used for controlling the resonances and Sierpinski carpet is developed for matching.

Thermally and optically tunable sub-terahertz superconducting fishnet metamaterial

In this paper, a novel fishnet metamaterial structure is designed and analyzed under different material combinations and under different active controlling techniques. The results indicate that, the proposed fishnet metamaterial has a single resonance with double negativity at 0.39 THz when quartz substrate and aluminum is utilized in the design. Moreover, when the metallic parts are replaced with YBCO, the proposed design also exhibits double negativity with a stronger resonance and can be used as a switch between the double negative and single negative modes if the temperature is altered. In addition to these, when substrate (quartz) is replaced with MgO, the resonance shifts from 0.39 THz to 0.26 THz and shows double negativity. Moreover, switching properties under illumination can also be obtained when the silicon is utilized in the design (MgO-YBCO combination). According to these results, it is found that, in the case that the conductivity of silicon exceeds a certain value, the character of the resonance changes from double negative to the single negative mode.

Murine CD8+ Invariant Natural Killer T Cells are Negatively Selected by CD1d Expressed on Thymic Epithelial Cells and Dendritic Cells

ABSTRACTBackground: CD1d-dependent invariant natural killer (iNKT) cells are found as either CD4 single positive (SP) or CD4/CD8 double negative (DN) cells in mice. The size of the CD8+ iNKT population is extremely small. It is known that CD1d expression on developing thymocytes is sufficient for iNKT development and co-receptor choice, which is driven by Th-POK expression. This study aimed to examine the factors involved in the CD4/CD8 co-receptor choice of iNKT cells in addition to Th-POK-driven silencing of CD8 expression. Methods: In this study, we compared iNKT cells of wild-type (WT) mice with those of transgenic mice in which CD1d expression is restricted to developing thymocytes by the proximal Lck (pLCK) promoter. CD8 positive iNKT cell population were analyzed by flow cytometry. Results: We found that there was a substantial population of CD8+ iNKT cells in the thymus and spleen of transgenic mice, and these cells are negatively selected in between Stage 2 and Stage 3 of their developmental program by the CD1d expressed on Thymic epithelial cell (TEC) and Dendritic cells in WT mice. Conclusion: We conclude that TEC expression of CD1d in the murine thymus contributed to co-receptor choice of iNKT cells, in addition to Th-POK-driven silencing of CD8. Therefore, mostly CD4 SP and DN iNKT cells are produced under normal physiological conditions in mice.

Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity.

BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27-IgD- double-negative (DN) B cells, but not CD27-IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines.