Double-positive Research Articles

B-Raf is required for positive selection and survival of DP cells, but not for negative selection of SP cells

The duration of signaling through the MAP kinase (or ERK pathway) cascade has been implicated in thymic development, particularly positive and negative selection. In T cells, two isoforms of the MAP kinase kinase kinase Raf function to transmit signals from the T-cell receptor to ERK: C-Raf and B-Raf. In this study, we conditionally ablated B-Raf expression within thymocytes to assess the effects on ERK activation and thymocyte development. The complete loss of B-Raf is accompanied by a dramatic loss of ERK activation in both the double positive (DP) and single positive (SP) thymocytes, as well as peripheral splenocytes. There was a significant decrease in the cellularity of KO thymi, largely due to a loss of pre-selected DP cells, a decrease in DP cells undergoing positive selection, and a defect in SP maturation. B-Raf plays significant roles in survival of DP thymocytes and function of SP cells in the periphery. Surprisingly, we saw no effect of B-Raf deficiency on negative selection of autoreactive SP thymocytes, despite the greatly reduced ERK activation in these cells.

Differential effects of γc cytokines on postselection differentiation of CD8 thymocytes

AbstractThe primary consequence of positive selection is to render thymocytes responsive to cytokines and chemokines expressed in the thymic medulla. In the present study, our main objective was to discover which cytokines could support the differentiation of positively selected thymocytes. To this end, we have developed an in vitro model suitable for high-throughput analyses of positive selection and CD8 T-cell differentiation. The model involves coculture of TCRhiCD5intCD69− double-positive (DP) thymocytes with peptide-pulsed OP9 cells and γc-cytokines. We report that IL-4, IL-7, and IL-21 have nonredundant effects on positively selected DP thymocytes. IL-7 signaling phosphorylates STAT5 and ERK; induces Foxo1, Klf2, and S1pr1; and supports the differentiation of classic CD8 T cells. IL-4 activates STAT6 and ERK and supports the differentiation of CD8intPD-L1hiCD44hiEOMES+ innate CD8 T cells. IL-21 is produced by thymic epithelial cells and the IL-21 receptor-α is strongly induced on DP thymocytes undergoing positive selection. IL-21 signaling phosphorylates STAT3 and STAT5, but not ERK, and does not support CD8 T-cell differentiation. However, IL-21 has a unique ability to up-regulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Our data suggest that injection of recombinant IL-21 might enhance thymic output in subjects with age- or disease-related thymic atrophy.

Thymocyte apoptosis and proliferation modeling during rat thymic involution is influenced by ovarian hormones in a thymocyte subset-specific manner

The study was aimed to define the putative role of ovarian hormones in shaping thymocyte apoptosis and proliferation during thymic involution. Thymocytes from young adult and middle-aged rats ovariectomized (Ox) before puberty were examined for apoptosis and proliferation. Apoptosis and proliferation were measured in fresh thymocyte suspensions and in their 18-hour cultures, and fresh thymocyte suspensions, respectively. The thymocyte population and the major thymocyte subsets were analyzed following triple staining using anti-CD4 and anti-CD8 monoclonal antibodies and 7-AAD to label apoptotic or proliferating cells. The frequency of apoptotic cells was lower in thymocyte suspensions and cultures from Ox rats of both ages. This reflected in a diminished frequency of apoptotic cells amongst CD4+CD8+ double positive (DP) and CD4+CD8- single positive (SP), and DP cells in young and middle-aged Ox rats, respectively. Additionally, in thymocyte cultures from Ox rats the frequency of apoptotic cells amongst CD4+CD8- and CD4-CD8+ SP cells decreased with age, but increased within DP and CD4-CD8- double negative (DN) subsets, reaching in the former subset from middle-aged Ox rats higher values than in age-matched controls. The frequency of proliferating cells was also lower in Ox rats than in controls. This reflected the lower frequency of cycling cells amongst CD4+CD8- SP and CD4-CD8+ SP thymocytes in young rats, and DP and CD4-CD8+ SP thymocytes in middle-aged rats. Besides, in both SP and DP thymocyte subsets from Ox rats the frequency of proliferating cells declined with age. In conclusion, thymocyte apoptosis and proliferation exhibit ovarian hormone-dependent thymocyte subset specific alterations during thymic involution.

Plxnd1 Expression in Thymocytes Regulates Their Intrathymic Migration While That in Thymic Endothelium Impacts Medullary Topology

An important role for plexinD1 in thymic development is inferred from studies of germline Plxnd1 knockout (KO) mice where mislocalized CD69+ thymocytes as well as ectopic thymic subcapsular medullary structures were observed. Given embryonic lethality of the Plxnd1−/− genotype, fetal liver transplantation was employed in these prior analyses. Such embryonic hematopoietic reconstitution may have transferred Plxnd1 KO endothelial and/or epithelial stem cells in addition to Plxnd1 KO lymphoid progenitors, thereby contributing to that phenotype. Here we use Plxnd1flox/flox mice crossed to pLck-Cre, pKeratin14-Cre, or pTek-Cre transgenic animals to create cell-type specific conditional knockout (CKO) lines involving thymocytes (D1ThyCKO), thymic epithelium (D1EpCKO), and thymic endothelium (D1EnCKO), respectively. These CKOs allowed us to directly assess the role of plexinD1 in each lineage. Loss of plexinD1 expression on double positive (DP) thymocytes leads to their aberrant migration and cortical retention after TCR-mediated positive selection. In contrast, ectopic medulla formation is a consequence of loss of plexinD1 expression on endothelial cells, in turn linked to dysregulation of thymic angiogenesis. D1EpCKO thymi manifest neither abnormality. Collectively, our findings underscore the non-redundant roles for plexinD1 on thymocytes and endothelium, including the dynamic nature of medulla formation resulting from crosstalk between these thymic cellular components.

Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells

Antigen-specific Tcells represent a potential therapeutic avenue for a variety of conditions, but current approaches for generating such cells for therapeutic purposes are limited. In this study, we established iPSCs from mature cytotoxic Tcells specific for the melanoma epitope MART-1. When cocultured with OP9/DLL1 cells, these iPSCs efficiently generated TCRβ(+)CD4(+)CD8(+) double positive (DP) cells expressing a Tcell receptor (TCR) specific for the MART-1 epitope. Stimulation of these DP cells with anti-CD3 antibody generated a large number of CD8(+) Tcells, and more than 90% of the resulting cells were specific for the original MART-1 epitope. Stimulation of the CD8(+) Tcells with MART-1 antigen-presenting cells led to the secretion of IFNγ, demonstrating their specific reactivity. The present study therefore illustrates an approach for cloning and expanding functional antigen-specific CD8(+) Tcells that might be applicable in cell-based therapy of cancer.

Strict Major Histocompatibility Complex Molecule Class-Specific Binding by Co-Receptors Enforces MHC-Restricted αβ TCR Recognition during T Lineage Subset Commitment

Since the discovery of co-receptor dependent αβTCR recognition, considerable effort has been spent on elucidating the basis of CD4 and CD8 lineage commitment in the thymus. The latter is responsible for generating mature CD4 helper and CD8αβ cytotoxic T cell subsets. Although CD4+ and CD8+ T cell recognition of peptide antigens is known to be MHC class II- and MHC class I-restricted, respectively, the mechanism of single positive (SP) thymocyte lineage commitment from bipotential double-positive (DP) progenitors is not fully elucidated. Classical models to explain thymic CD4 vs. CD8 fate determination have included a stochastic selection model or instructional models. The latter are based either on strength of signal or duration of signal impacting fate. More recently, differential co-receptor gene imprinting has been shown to be involved in expression of transcription factors impacting cytotoxic T cell development. Here, we address commitment from a structural perspective, focusing on the nature of co-receptor binding to MHC molecules. By surveying 58 MHC class II and 224 MHC class I crystal structures in the Protein Data Bank, it becomes clear that CD4 cannot bind to MHC I molecules, nor can CD8αβ or CD8αα bind to MHC II molecules. Given that the co-receptor delivers Lck to phosphorylate exposed CD3 ITAMs within a peptide/MHC (pMHC)-ligated TCR complex to initiate cell signaling, this strict co-receptor recognition fosters MHC class-restricted SP thymocyte lineage commitment at the DP stage even though both co-receptors are expressed on a single cell. In short, the binding preference of an αβTCR for a peptide complexed with an MHC molecule dictates which co-receptor subsequently binds, thereby supporting development of that subset lineage. How function within the lineage is linked further to biopotential fate determination is discussed.

Experimental studies of slow wave based on the surface waves in a two-dimensional metamaterials waveguide

In this paper, we study the slow propagation characteristics of surface electromagnetic waves according to theoretical analysis in the MNG (μ-negative)-DPS (double-positive)-MNG waveguide, which is composed of two-dimensional (2D) metamaterials. The dispersion curves of this waveguide vary with circuit parameter (unit capacitors) of MNG metamaterial. At the cutoff frequency of the dispersion curve the group velocity of electromagnetic wave is equal to zero in theory. So we can obtain slow propagation of the surface waves at different cutoff frequencies just by changing the unit capacitor of the MNG metamaterial. Theoretical analysis also shows that the step changes in slow waves are obtained at some fixed frequencies through changing the unit circuit parameters. The experimental results are in good agreement with simulation results.

The majority of T cells, including Treg cells, are developed from CD4−CD8− T progenitor cells without the involvement of the CD4+CD8+ stage in the thymus

In this study, we examined CD4−CD8− (DN) cells as precursors of T cells. We provided evidence that DN-CD25+ T cells could express the gene encoding forkhead box P3 (Foxp3) at high levels but were unable to express genes for T cell receptor (TCR) and cluster of differentiation 3 (CD3). In contrast, DN-CD25− cells could express and produce TCR and CD3 but not Foxp3. Furthermore, DN cells exhibited a higher-level expression of cell surface marker CD25, as well as Foxp3, but reduced expression of CD44, CD3, and TCR when compared to CD4+CD8− T cells. In the process of T cell development in the thymus, we noted that CD25 was down-regulated, and there occurred an up-regulation of TCR during maturation of T cells. These results suggested to us that DN cells might represent precursors of Treg cells, which contradicts the traditional view that double-positive (DP) cells constitute an intermediate cell stage. In addition, studies from other groups demonstrated that DP cells were not required for gene expression involving major histocompatability complex, Notch protein that dictates fate of a cell during development, TCR, and recombination activating (RAG) genes, which are involved in B cell development. Consequently, it is not surprising that DP cells might not be involved in the process of T cell development. One may speculate that DP cells are in a state of degeneration with down-regulated cell surface markers, which would most likely die by apoptosis.

Size-Independent Cylindrical Resonator Half-Filled With DNG Metamaterial and Excited by a Line Source

A circular cylindrical resonator with metallic (PEC) walls is half-filled with double-negative (DNG) metamaterial that is anti-isorefractive to the double-positive (DPS) material filling the remaining volume of the resonator. The resonance condition of the structure is studied, and it is shown that the resonator may perform independently of diameter size. The problem of an electric line source parallel to the axis and located anywhere inside the DPS region is solved exactly. Numerical results are presented and discussed .

Size-Independent Cylindrical Wheel Resonator

A metallic cavity resonator in the shape of a circular cylinder is considered in the phasor domain. The resonator is filled with an even number of geometrically equal wedge-shaped sectors whose common edge is the cylinder axis. The sectors contain alternating double-positive (DPS) and double-negative (DNG) materials that are isoimpedance and anti-isorefractive to each other. It is shown that resonance occurs always, hence the resonator can be miniaturized.

CD44 and CD27 expression pattern in B cell precursor acute lymphoblastic leukemia and its clinical significance

The expression of CD44 and CD27 molecules correlates with the differentiation stage of B cell precursors. The present study was designed to investigate the prognostic relevance of CD44 and CD27 molecules in patients with B cell acute lymphoblastic leukemia (ALL). CD27 and CD44 expression was determined in 58 patients by flow cytometry and their relation to established prognostic factors and response to therapy was investigated. Four patterns of expression were found; CD27 single positive (SP) in 20.7 % of patients, CD44SP in 25.8 %, CD27CD44 double positive (DP) in 20.7 %, and CD27CD44 double negative (DN) in 32.8 %. CD27 expression and the CD27SP pattern correlated directly with TEL/AML1 genotype (P = 0.012). Conversely, CD44 expression and the CD44SP pattern correlated inversely with this genotype (P = 0.016). Patients with the DP pattern had a lower WBC count (P = 0.03), lower percentage of blasts in their bone marrow (P = 0.028), and higher platelet count, whereas CD44SP patients had a higher WBC count and higher percentage of bone marrow blasts. Moreover, a negative association between DN pattern and complete remission (CR) rate was detected (P = 0.03). Mean CD27 expression was significantly higher in low-risk group and in patients who achieved CR (P = 0.001), and in those with a higher platelet number (P = 0.046) and less extramedullary involvement (P = 0.008). Although survival and CR duration were longer in patients with DP pattern and shorter in those with DN pattern, the result did not reach statistical significance. The expression of CD27 together with CD44 showed a relationship with several established risk factors as well as response to therapy, indicating the biological significance of these molecules in ALL.

Reducing radar cross section by investigation electromagnetic materials

Decreasing the Radar Cross Section (RCS) is investigated in electromagnetic materials, i.e. double-positive (DPS) , double-negative (DNG) , epsilon-negative (ENG) and mu-negative (MNG) materials. The interesting properties of these materials lead to a great flexibility in manufacturing structures with unusual electromagnetic characteristics. The valid conditions for achieving the transparency and gaining resonance for an electrically small cylinder are established, in this corresponding The effect of incidence direction on RCS inclusive of transparency and resonance conditions is also explored ,through computer simulations for an electrically small cylinder.

Role of OGR1 in myeloid-derived cells in prostate cancer

Ovarian cancer G-protein-coupled-receptor-1 (OGR1) is a tumor metastasis suppressor in prostate cancer (PCa). OGR1 knockout mice (ogr1(-/-)) are grossly normal under physiological conditions, however, reduced melanoma tumorigenesis has been observed, with the mechanisms of this reduction completely unknown. In this work, we demonstrated that OGR1 deficiency in host cells significantly reduced tumorigenesis of PCa in mice. Adoptive transfer of WT CD11b(+) Gr1(+) double positive (DP) cells, but not T cells, was sufficient to allow tumor development in ogr1(-/-) mice. The expression of an M1 macrophage marker, inducible nitric oxide synthase (iNOS) was higher and expression of an M2 macrophage marker, arginase-1 (Arg 1) was lower in tumors from ogr1(-/-) mice compared with WT mice. Furthermore, coinjection of transgenic adenocarcinoma mouse prostate (TRAMP)-C2 cells with WT, but not ogr1(-/-) macrophages, increased tumor incidence in ogr1(-/-) mice. T-cell depletion experiments suggested that T cells were required for tumor rejection in ogr1(-/-)mice, although OGR1 expression in T cells may not be necessary. In summary, the expression of OGR1 in myeloid-derived cells, especially in DP cells, was required for PCa tumor cell-induced immunosuppression.

Glycerol‐3‐Phosphate Acyltransferase‐1 Gene Ablation Results in Altered Thymocyte Lipid Content and Reduces Thymic T Cell Production in Mice

Glycerol 3-phosphate acyltransferase-1 (GPAT-1) catalyzes the initial and rate-limiting step in de novo glycerophospholipid and triacylglycerol (TAG) biosynthesis. We have previously shown that peripheral T cell proliferation and cytokine production is altered in GPAT-1 gene-ablated (KO) mice. This finding is important in light of the reduction in GPAT-1 activity associated with aged T cells. To determine if the mechanism for altered peripheral T cell function is linked to altered T cell development, we assessed thymic function in 3, 6 and 16-week old GPAT-1 KO compared to wild type (WT) mice. At 16 weeks of age, there was a significant reduction in thymic T cell production in KO compared to WT mice but not at 6 weeks of age. The reduced thymic T cell production was associated with altered thymic development as confirmed by increased numbers of double-negative (DN) thymocytes and a significant reduction in the double positive (DP) thymocytes suggesting a developmental block at the DN stage. This change was accompanied by an increase in the single positive CD4 subset. These changes were associated with reduced glycerophospholipid mass while thymic cortex and medulla architecture was not altered by GPAT-1 KO. Taken together, these data suggest that GPAT-1 deletion is capable of reducing the number of new T cells produced via alterations in membrane receptor function rather than by causing deleterious changes within the thymic microenvironment explaining in part the observed alterations in peripheral T cell function.

The Levels of a G-CSF-Inducible pSTAT3+pSTAT5+ Subpopulation of MDS Progenitors with Leukemic Stem Cell Phenotype Predict the Response to Azacytidine

AbstractAbstract 3795Both basal and cytokine-induced phosho-STAT3 and pSTAT5 levels are altered in leukemias and profiling the STAT signaling network at the single cell level provides prognostic information (Irish et al, 2004). As cell signaling interacts with the epigenome (Mohammad et al, 2010) and numerous genes involved in signaling are aberrantly methylated in MDS (Figueroa et al, 2010) we investigated the alterations of STAT3/5 signaling in MDS progenitors during azacitidine therapy.Bone marrow samples of 58 high-risk MDS and AML/MDS patients were obtained before (d0) and 15 days (d15) after azacytidine initiation and at the indicated time points. According to the IWG response criteria, patients were divided into 4 groups: response (CR and PR, n=11 and n=5, respectively), hematologic improvement (HI, n=8), stable disease (SD, n=11) and failure (F, n=23). Mononuclear cells were either left untreated or stimulated with G-CSF and GM-CSF for 15′ and then stained intracellularly with the indicated antibodies. The hematopoietic hierarchy and G-CSF receptor (CFS3R) transcripts by qPCR were assessed on immunomagnetically purified Lin-CD34+ and CD34+ cells, respectively. Statistical comparisons were done by ANOVA and unpaired or paired t-tests as appropriate. STAT signaling profiles (SPs) in CD34+ cells were grouped using complete linkage hierarchical clustering and correlated with treatment response, karyotype and transfusion rate by using χ2 or Fisher Exact tests.Unsupervised clustering of pretreatment SPs in CD34+ cells identified 2 clusters (Fig 1A) mainly differing in the degree of potentiated STAT3/5 phosphorylation. Patients in cluster I displayed weak potentiated expression of pSTAT3/5 and had marginally better response to azacytidine compared to the ones in cluster II (p=0.06), whereas there were no differences among the two groups regarding the MDS subtype (p=0.41), karyotype (p=0.45) and transfusion rate (p=0.33). We further identified a G-CSF-inducible pSTAT3/5 double positive (DP) subpopulation of MDS CD34+ cells, whose levels both at d0 and d15 of the 1st cycle were inversely associated with response (Fig 1B), whereas its kinetics were following the disease course and response to azacytidine (Fig 1C,D). The DP subset was enriched in GMPs compared to the G-CSF-unresponsive pSTAT3/5 double negative (DN) subpopulation, suggesting a higher leukemia initiating cell activity (Goardon et al, 2011), whereas the DN subset was marginally enriched in MEPs (Fig 2). Also, compared to the DN subset, the DP population exhibited decreased Ki67 expression and increased Bcl2 and p53 levels (Fig 3A), indicating quiescence and increased antiapoptotic and tumor suppressive properties, respectively. Of note, the levels of the above molecules in the two subsets remained unaltered both at d0 and d15, indicating that these subsets represent genuine cellular entities with solid properties. To determine if CSF3R expression and/or its modulation by azacitidine are responsible for the alterations of the DP population, we checked CSF3R levels in isolated CD34+ cells from patients with either absence or full expression of the DP subset because CSF3R is downregulated after G-CSF stimulation. The two groups showed identical protein and mRNA levels of CSF3R at both d0 and d15 (Fig 3B,C).In summary, we identified a G-CSF-inducible pSTAT3/5 DP subpopulation with leukemic stem cell properties, which is potentially involved in MDS biology and epigenetically modulated, as implied by its kinetics during hypomethylating therapy. More important, given the strong association of pretreatment levels of the DP subset with the response to azacitidine, the latter subset may serve both as a treatment target and response biomarker. [Display omitted] [Display omitted] [Display omitted] Disclosures:Kotsianidis:Genesis-Pharma: Honoraria, Research Funding.

FLVCR, a Heme Exporter, Is Required for Peripheral T Cell Survival.

AbstractAbstract 2162Heme is essential for every mammalian cell, however, free heme can induce free radical formation and cellular damage, therefore cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Conditional deletion of Flvcr was shown to cause progressive anemia in neonatal and adult mice (Science 319:825–8, 2008). Using a transplant model, we previously demonstrated that Flvcr-deleted thymocytes were blocked at the CD4+CD8+ double-positive (DP) stage (Blood [ASH Annual Meeting Abstracts] 114: 913, 2009). To characterize the temporal requirement for FLVCR in developing thymocytes, we crossed Flvcrflox/flox mice to thymocyte-specific cre recombinase strains: Lck-cre mice, which express cre in early CD4+CD8+ double-negative (DN) thymocytes, and CD4-cre mice, which turn on cre in late DN/early DP thymocytes. Flvcrflox/flox;Lck-cre mice had similar numbers of DN and DP thymocytes compared to controls, however, CD4+ and CD8+ single-positive (SP) thymocytes and peripheral T cells were nearly absent, similar to what we observed in our previous transplant model. In contrast, Flvcrflox/flox;CD4-cre mice had intact thymic development with normal numbers of SP, but there were few CD4+ and CD8+ T cells in the periphery. When we analyzed deletion efficiency of these T cells, CD8+ T cells showed only 50% Flvcr deletion and were nearly all CD44-high, implying that only incompletely-deleted CD8+ T cells survived and expanded. Taken together, these results show that FLVCR is required not only for T cell development beyond the DP stage, but also for the survival of mature T cells in the periphery.We next adoptively transferred thymocytes from Flvcrflox/flox;CD4-cre mice or controls into sub-lethally irradiated Rag1−/− mice. Normal SP thymocytes undergo homeostatic proliferation when transferred into an “empty” host. At day 12 and 20 post-adoptive transfer, few Flvcrflox/flox;CD4-cre CD4+ or CD8+ T cells were found, in contrast to mice that had received Flvcr+/flox;CD4-cre thymocytes. To determine whether Flvcr-deleted T cells failed to undergo homeostatic proliferation, we used carboxyfluorescein succinimidyl ester (CFSE) to label Flvcrflox/flox;CD4-cre or control thymocytes prior to adoptive transfer. At day 8, similar numbers of Flvcrflox/flox;CD4-cre and control T cell were found in the periphery and both had diluted CFSE equally, thus initial proliferation was not affected. However, by day 20, few Flvcr-deleted T cells were present compared to controls. Experiments are currently underway to understand how and why Flvcr-deleted T cells fail to persist long-term.The finding that FLVCR is required for T cell development and peripheral survival is intriguing because there is no known specific role for heme in T cell development or function. We carried out transcriptional profiling on sorted DP thymocytes from Rag1−/− mice transplanted with Flvcr-deleted or control bone marrow to determine whether FLVCR loss led to gene expression changes that might explain the block in T cell development. Surprisingly, there were few transcriptional changes, suggesting that FLVCR loss has an abrupt impact on T cell development late in the DP stage. This finding, together with the apparent normal development of Flvcr-deleted B lymphocytes and myeloid lineages, leads us to hypothesize that FLVCR plays a specific role in T cell development starting at the DP stage and persisting throughout T cell life. FLVCR is a member of the major facilitator superfamily of secondary active transporters. While FLVCR has been shown to export heme, it is not known whether it can import or export other small molecules or metabolites. We are now using a bioinformatics approach on published datasets to analyze metabolic gene expression during normal thymic development and in various mature T cell subsets to identify metabolic pathways that are specific for the DP-SP transition in thymocytes as well as in mature, peripheral T cells. We will then test whether these pathways are altered in Flvcr-deleted thymocytes and mature T cells. These studies may uncover a new role for heme in T cell metabolism, function, and survival, or a new non-heme role for FLVCR. Disclosures:No relevant conflicts of interest to declare.

Innate Lymphoid Cell-Derived IL-22 Regulates Epithelial Recovery From Gvhd

Abstract 339There is little understanding of the maintenance and regeneration of epithelial tissues after allogeneic transplant. Most clinical strategies to limit epithelial damage from graft vs. host disease (GVHD) also limit post-transplant immune function. Damage to the gastrointestinal (GI) tract from GVHD is a major cause of morbidity and mortality, and damage to the thymus from pre-transplant conditioning and GVHD can impair immune reconstitution, predispose patients to infection, and increase the risk of relapse. Therefore, understanding of tissue damage and recovery could lead to strategies selectively protecting epithelial tissues, improving intestinal barrier function, and promoting immune reconstitution without worsening post-transplant immunosuppression.We have recently identified that IL-22 from recipient-derived innate lymphoid cells (ILC) is critical for promoting intestinal recovery from GVHD and for promoting thymic recovery from radiation/pre-transplant conditioning. IL-22 deficient mice demonstrated significantly reduced thymopoiesis after total body irradiation (TBI), and IL-22 deficient murine bone marrow transplant (BMT) recipients demonstrated increased GVHD mortality and intestinal histopathology, deficiency of the antimicrobial molecules Reg3γ and Reg3β, and loss of intestinal stem cells needed for epithelial recovery. The source of thymic and intestinal IL-22 was RORγ+CD3−NKp46−IL-7R+CCR6+ lymphoid-tissue-inducer-like cells. Similar to as had been observed in the thymus, intestinal ILC produced IL-22 in response to IL-23, which was upregulated after TBI (p<.05 small intestine, p<.001 large intestine). IL-22 was also upregulated in response to TBI, but not in p40-deficient mice lacking IL-23 (p<.05 small intestine, p<.01 large intestine). ILC were radioresistant, as lethal TBI led to a three-fold increase in the intestinal ILC:CD4 ratio (p<.05). Furthermore, recipient-derived ILC comprised more than 50% of intestinal lamina propria ILC three months after T cell-depleted BMT, well after donor myeloid reconstitution and after donor reconstitution of the intestinal T cell compartment as well (Figure 1).Although intestinal ILC could survive lethal TBI, they were significantly depleted by both MHC mismatched (B6BALB/c) and MHC matched (LPB6) GVHD. Similarly, GVHD led to depletion of thymic IL-22+ ILC and reduction in thymic IL-22 levels (p<.001). Thymic IL-22 was critical for maintaining thymopoiesis during GVHD, as IL-22 deficient BMT recipients demonstrated significantly greater loss of double positive (DP) thymocytes after MHC-mismatched BMT. We previously identified that IL-21 receptor (IL-21R) signaling contributes to the migration of alloreactive donor T cells to the GI tract and that IL-21R-deficent donor T cells mediate significantly reduced GI GVHD. Given the similar homing molecules involved in the migration of donor T cells to the GI tract and thymus in GVHD, we evaluated the role of IL-21 in thymic GVHD. Donor T cell IL-21R deficiency led to increased thymopoiesis and DP thymocytes (p<.001), but not in IL-22-deficient recipients. ILC evaluation indicated that this IL-22 dependency was because IL-21R-deficiencient donor T cells had a reduced capacity to eliminate thymic ILC during GVHD (Figure 2). Therefore, donor T cell IL-21 signaling was critical for the elimination of recipient thymic ILC during GVHD, and preservation of the ILC compartment allowed for the IL-22 mediated regeneration of thymopoiesis. Finally, we also found that administration of rIL-22 post-BMT could reverse the thymic damage caused by GVHD and elimination of ILC, restoring the numbers of DP thymocytes to a level similar to what was observed after T cell-depleted BMT.In summary, IL-22+ ILC are radioresistant and capable of regulating tissue-specific epithelial recovery after allogeneic BMT. However, recipient ILC are extremely sensitive to GVHD, leading to a loss of the IL-22-mediated recovery response. IL-21 blockade can prevent the elimination of recipient thymic ILC by donor T cells in GVHD, and IL-22 administration can restore the thymopoiesis that is lost in GVHD due to ILC elimination. Maintenance of epithelial function post-BMT is thus an active innate immune response requiring cooperation between both recipient stroma and recipient hematopoietic cells. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The TAL1 Complex Represses the FBXW7 Tumor Suppressor Through Mir-223 in Human T-Cell Acute Lymphoblastic Leukemia

Abstract 1296The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL) and causes T-ALL in murine transgenic models, emphasizing its importance in the molecular pathogenesis of this disease. However, the mechanism by which TAL1 leads to transformation of thymocytes is unclear. Dysregulation of miRNAs play an important role in tumorigenesis in diverse cancer types. A recent study identified miR-223 as the most abundant miRNA in T-ALL patient samples and was oncogenic by virtue of its ability to accelerate Notch-induced T-ALL in a murine model (Mavrakis et al. Nature Genetics 2011). However, the underlying mechanisms leading to dysregulated miRNA expression in T-ALL remain poorly understood.In order to explore the hypothesis that aberrant expression of miRNAs is mediated by the TAL1 oncogene in T-ALL, we generated high-resolution maps of the genome-wide occupancy of the TAL1 complex, including E2A, HEB, GATA3, LMO2 and RUNX1 by chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq). Analysis of binding sites in two TAL1-positive T-ALL cell lines (Jurkat and CCRF-CEM cells) and two primary T-ALL samples identified 54 miRNAs where binding of the TAL1 complex was within 10 kb of either the transcriptional start sites or the start sites of genes that contain miRNAs in their intronic regions. To determine which of these miRNAs were not only directly bound, but also regulated by the TAL1 complex, we analyzed global changes in miRNAs after knockdown of TAL1 in Jurkat cells using two independent shRNAs. By miRNA expression profiling, we identified significant changes in expression of 25 miRNAs, of which nine were down-regulated on TAL1 knockdown (and thus positively regulated by TAL1) and 16 were up-regulated on TAL1 knockdown (and thus negatively regulated by TAL1). Of these 25 miRNAs, four (miR-223, miR181a*, miR-26a and miR-29c) were shown to be direct targets of the TAL1 complex based on our ChIP-seq data.We chose to focus on miR-223 because it exhibited the most dynamic down-regulation after TAL1 knockdown. ChIP-qPCR validated binding of the TAL1 complex to a region within 4 kb of the miR-223 transcriptional start site. Analysis of RNA polymerase II and CBP binding showed significant enrichment, and high levels of H3K4M3 and H3K79M2 modification were detected indicative of transcriptional initiation and elongation of this locus. Furthermore, expression of miR-223 was significantly higher in the TAL1-positive cell lines (n=13) as compared to the TAL1-low cells (n=10) (P<0.0001). miR-223 levels also closely mirrored TAL1 levels in murine thymic subsets, with marked down-regulation after the DN2 stage, suggesting miR-223 is a physiological target of TAL1 during normal thymic development, and that its overexpression in TAL1-positive T-ALL cells, arrested at the double-positive (DP) stage, is aberrant compared to their normal DP counterpart.To test the hypothesis that the growth inhibition observed after TAL1 knockdown is mediated by decreases in miR-223 expression, we retrovirally infected Jurkat and RPMI-8402 T-ALL cell lines with a miR-223 construct, such that miR-223 expression was no longer under the control of TAL1 in these cells. Forced expression of miR-223 partially rescued the growth inhibitory effects induced by TAL1 knockdown, in both a lentiviral and doxycycline-inducible shRNA system. Additionally, inhibition of mature miR-223 by lentiviral infection of a miR-223 shRNA construct led to significant growth inhibition of TAL1-positive cell lines through the induction of apoptosis. Thus, maintenance of miR-223 expression is required for optimal growth of TAL1-positive T-ALL cells.The highest ranked predicted target of miR-223 by Targetscan is the FBXW7 tumor suppressor, a ubiquitin ligase that is mutated in a significant proportion of T-ALL patients and targets oncogenes such as c-MYC, NOTCH and mTOR for degradation. Accordingly, overexpression of miR-223 in TAL1-low miR-223-low T-ALL cells markedly down-regulated FBXW7 protein expression. Furthermore, the up-regulation of FBXW7 protein expression observed on knockdown of TAL1 in TAL1-positive cell lines could be prevented by retroviral miR-223 expression. Thus, miR-223 is an important target of TAL1 and links the TAL1 oncogene to repression of the FBXW7 tumor suppressor. Disclosures:No relevant conflicts of interest to declare.

Phagocytosis Is A Mechanism of Action for Daratumumab

AbstractAbstract 4054Daratumumab (DARA) is a human IgG1 CD38 antibody with broad-spectrum killing activity. DARA induces killing of CD38-expressing tumor cells, including fresh cells from multiple myeloma (MM) patient samples, via diverse mechanisms. These prominently include the Fc-dependent effector mechanisms complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers et al. J. Immunol. 2011). In this study we show that DARA is also able to induce tumor cell killing via antibody-dependent cellular phagocytosis (ADCP) as an additional Fc-dependent effector mechanism.In a first set of experiments, we studied ADCP with human macrophages as effector cells. Calcein-AM-labeled Daudi tumor cells, a Burkitt's lymphoma cell line, were mixed with human macrophages in the absence or presence of DARA. Specific DARA-induced phagocytosis was analyzed in flow cytometry by measuring the percentage of calcein-AM+/CD11b+ double-positive (DP) macrophages. Both classical GM-CSF activated and alternative M-CSF activated macrophages mediated DARA-specific ADCP of the Burkitt's lymphoma cells. To further explore the in vivo contribution of ADCP in the mechanism of action (MoA) of DARA, we studied DARA mediated phagocytosis with murine macrophages. In vitro ADCP with M-CSF-stimulated bone marrow-derived murine macrophages showed a dose-dependent DARA-specific effect on the Burkitt's lymphoma cell lines Ramos and Daudi, resulting in up to 24% and 43% DP macrophages and a 25% and 50% tumor cell reduction, respectively. Furthermore, dose-dependent DARA-specific phagocytosis was observed with patient-derived MM cell lines L363 and UM9, which were transduced with CD38 to obtain levels CD38 expression as they are generally observed in primary MM patient samples. With life-cell imaging we found that ADCP of Daudi and Ramos cells occurred very rapid and efficiently. Interestingly, our recordings document that single macrophages could engage multiple target cells and that they were able to engulf up to six tumor cells sequentially in a 30 min period. This suggests that ADCP might be a very potent MoA of DARA in vivo, which we are currently studying in a mouse xenograft model.In conclusion, in addition to CDC and ADCC, we now show that DARA can also induce killing of CD38 expressing tumor cells via phagocytosis. This very fast and potent MoA might contribute to the treatment efficacy of DARA in hematological tumors, especially at sites where high numbers of macrophages reside, such as the bone marrow. Disclosures:Overdijk:Genmab BV: Employment. Verploegen:Genmab BV: Employment. Groen:Genmab BV: Research Funding. Martens:Genmab BV: Research Funding. Lammerts van Bueren:Genmab BV: Employment. Bleeker:Genmab BV: Employment. Parren:Genmab BV: Employment.

Microwave collimation based on zero index metamaterials with Dirac point

The microwave zero index metamaterials (ZIMs) can be realized by loading lumped elements into two-dimensional transmission lines (TLs) with Dirac cones whose linear dispersion can appear around the center of the Brillouin zone. Based on Snell's law, the refracted angle of waves nearly equals zero when a point source radiates from the ZIM TL into the double positive (DPS) one in the ZIM-DPS TL structure. Experimental results demonstrate that at Dirac point the curved wavefronts in the ZIM region are transformed into planar ones in DPS region.