Spleen Populations Research Articles

Ischemic postconditioning protects against focal cerebral ischemia by inhibiting brain inflammation while attenuating peripheral lymphopenia in mice

BackgroundIschemic postconditioning (IPostC) has been shown to attenuate brain injury in rat stroke models, but a mouse model has not been reported. This study establishes an IPostC model in mice and investigates how IPostC affects infiltration of leukocytes in the ischemic brain and lymphopenia associated with stroke-induced immunodepression. Material and methodsA total of 125 mice were used. IPostC was performed by a repeated series of brief occlusions of the middle cerebral artery (MCA) after reperfusion, in a focal ischemia model in mice. Infarct sizes, neurological scores, inflammatory brain cells and immune cell populations in lymph nodes, spleen and bone marrow were analyzed with fluorescence-activated cell sorting (FACS). ResultsIPostC performed immediately, 2min and 3h after reperfusion significantly reduced infarct sizes and attenuated neurological scores as measured up to 3days post-stroke. In the group with strongest protection, infarct sizes were reduced from 49.6±2.8% (n=16) to 27.9±2.9% (n=10, P<.001). The spared infarct areas were seen in the ischemic penumbra or ischemic margins, i.e., the border zones between the cortical territories of the anterior cerebral artery and those of the MCA, as well as in the ventromedial and dorsolateral striata. FACS analyses showed that IPostC significantly blocked increases in the numbers of microglia (CD45intCD11b+), macrophages (CD45hiCD68+), CD4 T cells (CD45+CD4+) and CD8 T cells (CD45+CD8+) as well as B lymphocytes (CD45+CD19+) in the ischemic brain (n=5/group). Reduced-immune cell numbers in the peripheral blood and spleen were increased by IPostC while immune cell populations in the bone marrow were not altered by IPostC. ConclusionsIPostC reduced brain infarction and mitigated neurological deficits in mice, likely by blocking infiltration of both innate and adaptive immune cells in the ischemic brain. In addition, IPostC robustly attenuated peripheral lymphopenia and thus improved systemic immunodepression.

In Vivo Knockdown of TAK1 Accelerates Bone Marrow Proliferation/Differentiation and Induces Systemic Inflammation

TAK1 (TGF-β Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-β genetic models, our data suggest that in our model the function of TAK1 in TGF-β signal-transduction is overruling its function in pro-inflammatory signaling.

Maternal Protein Deficiency during a Gastrointestinal Nematode Infection Alters Developmental Profile of Lymphocyte Populations and Selected Cytokines in Neonatal Mice

Neonatal immune development begins in pregnancy and continues into lactation and may be affected by maternal diet. We investigated the possibility that maternal protein deficiency (PD) during a chronic gastrointestinal (GI) nematode infection could impair neonatal immune development. Beginning on d 14 of pregnancy, mice were fed protein-sufficient (PS; 24%) or protein-deficient (PD; 6%) isoenergetic diets and were infected weekly with either 0 (sham) or 100 Heligmosomoides bakeri larvae. Pups were killed on d 2, 7, 14, and d 21 and dams on d 20 of lactation. Lymphoid organs were weighed. Cytokine concentration in maternal and pup serum and in milk from pup stomachs and lymphoid cell populations in pup spleen and thymus were determined using luminex and flow cytometry, respectively. GI nematode infection increased Th2 cytokines (IL-4, IL-5, IL-13), IL-2, IL-10, and eotaxin in serum of dams whereas PD reduced IL-4 and IL-13. The lower IL-13 in PD dams was associated with increased fecal egg output and worm burdens. Maternal PD increased vascular endothelial growth factor in pup milk and eotaxin in pup serum. Maternal infection increased eotaxin in pup serum. Evidence of impaired neonatal immune development included reduced lymphoid organ mass in pups associated with both maternal infection and PD and increased percentage of T cells and T:B cell ratio in the spleen associated with maternal PD. Findings suggest that increases in specific proinflammatory cytokines as a result of the combination of infection and dietary PD in dams can impair splenic immune development in offspring.

Emergence of Long-Lived Autoreactive Plasma Cells in the Spleen of ITP Patients Treated with Rituximab

AbstractAbstract 620 Introduction:The spleen in primary immune thrombocytopenia (ITP) is not only the major site of platelet destruction but is also the main site of auto-antibody production. Accordingly, splenectomy as a second-line treatment has resulted in a durable platelet response in two-thirds of the patients. New strategy, using anti-CD20-induced B-cell depletion (Rituximab, RTX), leads to 40–50% of short term and 20% of long-lasting response. Less is known about B-cell depletion in spleen and the nature of the resistant pathogenic cells. In this study, we analyzed at the population and single cell level the splenic antibody secreting cells, in healthy donors and active chronic ITP patients, treated or not with RTX. Results:We took advantage of these different therapeutic outcomes to analyze the splenic B cell compartment of ITP patients, either not treated with Rituximab (n=5), or treated (n=10) and presenting with a primary failure of the B-cell depletion therapy, and compared them with healthy donors (n=5). Plasma cells (that lack CD20 expression) were the major residual population in spleens from Rituximab-treated patients (0.5% of CD45+ spleen mononuclear cells; range 0.06–1%). A fraction of these plasma cells were autoreactive (0.5–1.2% of IgG secreting cells being anti-GpIIbIIIa specific), thus accounting for the persistence of the disease and the failure of RTX treatment. Using gene profiling and single cell multiplex quantitative RT-PCR (Fluidigm dynamic arrays), we showed that such Rituximab-resistant plasma cells display a unique long-lived transcriptional program, similar to the one of bone marrow plasma cells. In contrast, splenic PC from healthy donors and from untreated ITP patients displayed an intermediate gene expression profile between the ones of long-lived PC and proliferating plasmablasts, with the simultaneous expression of cell cycle genes and survival/anti-apoptotic factors. BAFF secretion was increased two-fold in RTX-spleen supernatants compared to ITP (P<0.05), and might selectively contribute to the survival of the resident RTX-PC population. Conclusion:Our results demonstrate that residual long-lived plasma cells, with a few percents of them secreting anti-GpIIbIIIa antibodies, are found in the spleen 3–6 months following RTX treatment, thus providing a possible explanation for the failure of therapy, and the success of subsequent splenectomy. As opposed to accepted schemes, our data suggest that the normal human spleen is not a niche for long-lived PC, and that auto-immune/inflammatory conditions do not create either a favorable environment for their persistence. We propose in contrast that it is the B-cell depletion itself and the specific cytokinic condition it provides that generates suitable environment for the maturation of long-lived plasma cells in the spleen. This observation suggests that interfering with the differentiation and/or survival of this RTX-induced long-lived plasma cell population could obviate the need for splenectomy in the case of RTX-treatment failure. Disclosures:Off Label Use: rituximab.

A Role for Interferon Regulatory Factor 4 in the Development of CLL

AbstractAbstract 714Chronic Lymphocytic Leukemia (CLL) representing 30% of all the adult leukemia is a heterogeneous B cell malignancy with complex clinical behavior and pathophysiology. Even though an increasing body of literature has helped in understanding the biology of CLL the precise molecular events that underlie the pathogenesis of this disease are still not fully understood. Recent Whole Genome Sequencing and Genome Wide Association Studies have linked different genetic loci to the origin and evolution of the disease. A Genome Wide Association Study (GWAS) by Di Bernardo et al identified Single Nucleotide Polymorphisms in the 3'UTR of Interferon Regulatory Factor 4 (IRF4) as a familial risk allele for CLL. These SNPs are associated with lower mRNA levels of IRF4 in individuals carrying the risk allele. Another study by Havelange V. et al identified heterozygous mutations in the DNA binding domain of IRF4 in patients with CLL. These studies have linked lower levels of IRF4 to the pathogenesis of CLL. Even though studies have linked low levels of IRF4 to the pathogenesis of CLL, whether low levels of IRF4 indeed contribute to CLL development remains to be determined.In rodent, CLL cells are presumably derived from B1 cells. Like CLL cells, B1 cells are B220+CD5+ and possess self-reactive B cell receptors (BCR). B1 cells normally reside in peritoneal and pleural cavities and constitute only about 5% of total B lymphocytes in mice. However, B1 cell population is dramatically elevated in the VH11 knock-in mice. VH11 family of variable IgH chain is expressed exclusively on B1 cells where it pairs with Vk9 to form BCR that recognizes phosphatidylcholine on senescent RBCs. We did not observe an overt increase of CLL development in IRF4 deficient mice. However, when we crossed the VH11 knock-in mice to an IRF4 deficient mice it resulted in a CLL like lymphoproliferative disorder with marked expansion of CD5+ B220low/int population in blood, spleen and other lymphoid organs. To monitor pathogenesis of CLL, we collected blood from saphenous vein of IRF4−/−VH11 and IRF4+/+VH11 littermate control mice monthly. A total of ten mice from each group were used for this study. The appearance of a monoclonal B220lowCD5+ population and its percentage among peripheral blood mononuclear cells (PBMC) were used for the initial CLL diagnosis. The diagnostic criterion for CLL in VH11 mice is the appearance of monoclonal B220lowCD5+ cells constituting over 20% of PBMCs. If the percentage of the B220lowCD5+ clone is under 20% of the PBMC, the diagnosis would be monoclonal B cell lymphocytosis (MBL), a disease which can progress into CLL. Interestingly, 6 out of 10 IRF4−/−VH11 mice developed CLL after just 5 months while the four remaining IRF4−/−VH11 mice developed MBL. In contrast, none of the IRF4+/+VH11 control mice developed CLL or MBL. Two of the mice developed aggressive CLL where leukemic cells infiltrated liver and other non-lymphoid organs. BrdU incorporation assay identified spleen as the primary site of CLL cells expansion although proliferating CLL cells were also identified in lymph nodes and bone marrow. We further isolated CLL cells from IRF4−/−VH11 mice and transplanted them into immunodeficient mice (Rag2−/−gamma−/−). Our result shows that the disease was transplantable as all the transplanted mice succumbed to CLL within 1 month post-transplantation. Treatment of IRF4−/−VH11 CLL cells with specific Syk and Btk inhibitors targeting BCR signaling showed that like human CLL, the leukemic cells were depended on active BCR signaling for their survival and proliferation.In summary, we provide evidence for a causal relationship between low levels of IRF4 and development of CLL. Further studies are needed to provide mechanistic insights into how IRF4 is involved in the development and progression of this disease. The fine dissection of this relationship will allow for development of new therapeutic strategies and identification of new prognostic markers for diagnosis and treatment of CLL. Disclosures:No relevant conflicts of interest to declare. Disclosures:No relevant conflicts of interest to declare.

Activation of Fms-Like Tyrosine Kinase 3 Signaling Enhances Survivin Expression in a Mouse Model of Rheumatoid Arthritis

Survivin is known as an inhibitor of apoptosis and a positive regulator of cell division. We have recently identified survivin as a predictor of joint destruction in patients with rheumatoid arthritis (RA). Flt3 ligand (Flt3L) is expressed in the inflamed joints and has adjuvant properties in arthritis. Studies on 90 RA patients (median age 60.5 years [range, 24–87], disease duration 10.5 years [range, 0–35]) show a strong positive association between the levels of survivin and Flt3L in blood. Here, we present experimental evidence connecting survivin and Flt3L signaling. Treatment of BALB/c mice with Flt3L led to an increase of survivin in the bone marrow and in splenic dendritic cells. Flt3L changed the profile of survivin splice variants, increasing transcription of the short survivin40 in the bone marrow. Treatment with an Flt3 inhibitor reduced total survivin expression in bone marrow and in the dendritic cell population in spleen. Inhibition of survivin transcription in mice, by shRNA lentiviral constructs, reduced the gene expression of Flt3L. We conclude that expression of survivin is a downstream event of Flt3 signaling, which serves as an essential mechanism supporting survival of leukocytes during their differentiation, and maturation of dendritic cells, in RA.

Cell Population in Spleens During Antibody-Mediated Rejection

In the treatment of refractory antibody-mediated rejection (AMR), splenectomy has been associated with surprisingly rapid recovery of renal function. The mechanism is still unclear. We review 11 recipients, who underwent rescue splenectomy (RS) as a treatment of AMR within 3 months after kidney transplantation. At transplantation, all patients had undergone desensitization for initially positive crossmatch to their prospective donors. The cellular populations of the spleen were analyzed by immunohistochemistry. For comparison, we obtained spleen specimens from eight controls who were non-transplantation patients. Rejection occurred in all the patients early after transplantation (mean [SD], 7.1 [5.7] days). One graft was lost 4 weeks after kidney transplantation. A significantly higher number of plasma cells (PCs) (P=0.049) and lower number of T and B lymphocytes (P=0.02 and P=0.005, respectively) were detected in the RS group compared with the control group. By analyzing the PC variations in the RS group, significantly lower numbers of PCs were detected in the spleens of patients who received rituximab before splenectomy (P=0.0004). In contrast, a higher number of PCs were found in patients (n=3) who did not respond to splenectomy and subsequently underwent bortezomib treatment and recovered their renal function (P=0.02). Splenectomy may reverse AMR by debulking PCs. Our analysis suggests that patients with a very high load of PCs may not be rescued by splenectomy alone and may need additional treatments.

Immunomodulatory effects of citrus fruit auraptene in vitro and in vivo

Effects of auraptene on immune cells in vitro and in vivo were examined. IgM production by human hybridoma HB4C5 cells was enhanced by auraptene. Auraptene also stimulated IgA and IgG production by mouse primary splenocytes, and IgA and IgM production by lymphocytes from mesenteric lymph nodes in vitro. To examine the effect of auraptene on immune system in vivo, auraptene was orally administered to mice for 14days. Immunoglobulin production by splenocytes and lymphocytes from mesenteric lymph nodes were significantly activated by intake of auraptene. Moreover, IL-4, IFN-γ, and TNF-α production by splenocytes was accelerated under concanavalin A-activated condition. B cell population in spleen was increased by auraptene intake.

Caprylic acid reduces Salmonella Enteritidis populations in various segments of digestive tract and internal organs of 3- and 6-week-old broiler chickens, therapeutically ,

We investigated the efficacy of feed supplemented with caprylic acid (CA), a natural, 8-carbon fatty acid for reducing Salmonella enterica serovar Enteritidis colonization in commercial broiler chickens. In separate 3- and 6-wk trials, 1-d-old straight-run broiler chicks (n = 70 birds/trial) were assigned to a control group (challenged with Salmonella Enteritidis, no CA) and 2 replicates of 0.7 and 1% CA (n = 14 birds/group). Water and feed were provided ad libitum. On d 1, birds were tested for any inherent Salmonella (n = 2 birds/group). For the 3-wk trial, on d 5, birds were challenged with 8 log(10) cfu of Salmonella Enteritidis of a 4-strain mixture by crop gavage, and after 5 d postchallenge, birds (n = 2 birds/group) were euthanized to ensure Salmonella Enteritidis colonization. Caprylic acid was supplemented the last 5 d before tissue collection (n = 10 birds/group). For the 6-wk trial, on d 25, birds were challenged and confirmed for Salmonella Enteritidis colonization. The birds (n = 10 birds/group) were euthanized for tissue samples after CA supplementation for the last 5 d. Caprylic acid at 0.7 or 1% decreased Salmonella Enteritidis populations in cecum, small intestine, cloaca, liver, and spleen in both 3- and 6-wk trials. Body weight of birds did not differ between the groups (P ≥ 0.05). Further, to elucidate a potential antibacterial mechanism of action of CA, we investigated if CA could reduce Salmonella Enteritidis invasion of an avian epithelial cell line and expression of invasion genes hilA and hilD. The cell invasion study revealed that CA reduced invasive abilities of all Salmonella Enteritidis strains by ~80% (P < 0.05). Gene expression studies indicated that CA downregulated (P < 0.001) Salmonella invasion genes hilA and hilD. These results suggest that supplementation of CA through feed could reduce Salmonella Enteritidis colonization in broiler chicken and potentially reduces the pathogen's ability to invade intestinal epithelial cells by downregulating key invasion genes, hilA and hilD.

&lt;i&gt;Lactobacillus acidophilus&lt;/i&gt; Strain L-92 Induces CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;+&lt;/sup&gt;Foxp3&lt;sup&gt;+&lt;/sup&gt; Regulatory T Cells and Suppresses Allergic Contact Dermatitis

The anti-allergic mechanism of heat-killed Lactobacillus acidophilus strain L-92 has not been fully investigated. Recent studies have reported that CD4(+)CD25(+)Foxp3(+) (forkhead box P3) T regulatory (Treg) cells play important roles in controlling allergic diseases. Hence, we examined the effect of orally administered L-92 on CD4(+)CD25(+)Foxp3(+) cell populations. BALB/c mice were supplemented daily with L-92 by gavage for 5 weeks. 2,4-Dinitrofluorobenzene (DNFB) was used to induce allergic contact dermatitis (ACD) in mice. Fluorescent-activated cell sorter (FACS) analysis was used to determine CD4(+)CD25(+)Foxp3(+) T cell populations in spleen and cervical lymph nodes (CLN). Interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and Foxp3 mRNA expressions in mouse ear skin were investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of CD4(+)CD25(+)Foxp3(+) T cell populations were significantly increased in both spleen and CLN of L-92-fed group than vehicle and control. In addition, L-92 produced higher levels of Foxp3, IL-10 and TGF-β compared to control mice. These results suggest that L-92 can up-regulate the number of Treg cells to suppress the progression of DNFB-induced contact dermatitis in mice.

Failure of Erythropoiesis and Megakaryocytopoiesis in RASA3 Mutant Scat Mice

Abstract 680Scat (severe combined anemia and thrombocytopenia) is a spontaneous, autosomal recessive mutation coisogenic with the BALB/cBy inbred mouse strain. Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. As its name implies, crisis episodes are characterized by severe anemia and thrombocytopenia, but significant lymphocyte depletion occurs as well. The first crisis episode begins in utero, lasts until postnatal day (P) 9 on average, and is associated with 10–15% mortality. Remarkably, in homozygotes that survive the first crisis, a remission phase occurs wherein the disease phenotype reverts to normal. This remission is transient, however, and is followed by a second crisis episode during which 94% of scat/scat mice die by P30. Previously we showed that the scat phenotype is transferrable via the hematopoietic stem cells and is also recapitulated in scat/scat, Hox11−/− double homozygotes in which a spleen does not develop, indicating that the splenic micro-environment plays little or no role in disease appearance or progression. Positional cloning of scat revealed a missense mutation in Rasa3 encoding a GTPase activating protein (GAP) that negatively regulates Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. We further showed that Rasa3 is a conserved gene critical to vertebrate erythropoiesis via morpholino knockdowns in zebrafish which resulted in profound anemia. Here we report data that shed further light on RASA3 function during hematopoiesis. Overall, the data indicate that defects in RASA3 profoundly and negatively impact erythropoiesis and megakaryocytopoieis through, at least in part, a Ras-mediated mechanism. FACS analyses of scat spleen and bone marrow erythroid populations reveal a severe block in erythropoiesis during crisis periods. In the spleen, despite an initial increase in size due to expansion of Ter-119+ cells, there is ultimately a loss of compensatory erythropoiesis resulting in a return to normal cellularity and a striking loss of hemoglobinized cells as the crisis phenotype deepens. In addition, the bone marrow shows loss of Ter-119+ cells and overall cell depletion during crisis. Megakaryocyte numbers are increased in scat crisis BM and spleen. By transmission electron microscopy, scat crisis megakaryocytes display features characteristic of a significant developmental delay: a disorganized demarcation membrane system with no platelet forming areas and few granules with hypersegmented nuclei and excess rough endoplasmic reticulum. In addition to the severe anemia and thrombocytopenia, a significant lymphopenia occurs in scat crisis mice. However, the scat phenotype is not lymphocyte mediated, as the scat phenotype is completely recapitulated in mice doubly homozygous for scat and the immunodeficient mutations, scid and Rag1tm1Mom, in which B- and T-lymphocytes are completely depleted. Together these results suggest that lymphopenia is a secondary phenomenon in scat, and the severe anemia and thrombocytopenia aspect of the phenotype neither follows from nor is dependent upon loss of lymphocytes. Despite the delay observed in erythroid differentiation, some mature red cells are produced although ∼50% of these are reticulocytes. By confocal microscopy, we show that RASA3 protein localizes to the plasma membrane as well as internal membrane compartments in wild type reticulocytes, where it partially colocalizes with CD71. Western blot analyses of reticulocytes after Percoll gradient purification show that RASA3 is lost during the maturation step, both in vivo and in vitro. Interestingly, in scat, RASA3 is present in reticulocytes, but appears to be mislocalized, the protein being found in the cytosol. Preparation of ghosts from wild type and scat reticulocytes confirms that RASA3 is not attached to the membrane in scat animals during crisis. In pull-down assays active GTP-bound Ras is increased in scat crisis reticulocytes when compared to wild type, suggesting that scat is a RASA3 loss of function mutation due to its mislocalization and demonstrating a critical role for the RASA3-Ras axis during mammalian erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Abstract 2603: Inhibition of Notch and mTOR by nelfinavir as a novel approach for T cell acute lymphoblastic leukemia (T-ALL)

Abstract Background: T-ALL accounts for 10-15% of childhood ALL and 25% of adult ALL. Although intensive chemotherapy improves disease free survival, patients who relapse or progress have a dismal prognosis. Notch1 is a promising target in T-ALL because it is commonly activated, and inhibitors of γ-secretase (GSI), an aspartic protease that processes Notch1, are in clinical trials. Nelfinavir is an HIV protease inhibitor with anti-cancer activity through its inhibition of the Akt/mTOR pathway as well as unknown mechanisms. Because the HIV protease is an aspartic protease, we hypothesized that a target for nelfinavir in cancer cells could be γ-secretase. Methods: Nelfinavir was compared to compound E, a commercially available GSI, in in vitro studies. Notch1 and mTOR signalling was evaluated in human and murine T-ALL cell lines by immunoblotting. γ-secretase activity was assessed in cell-free in vitro assays. Cell death was measured by flow cytometry after annexin V and propidium iodide staining. Nelfinavir was also tested in a transgenic model of T-ALL (established by overexpression of SCL and LMO1 in the mouse thymus) using microCT scans, flow cytometry and IHC. Results: Among 7 HIV protease inhibitors evaluated, only nelfinavir inhibited expression of the Notch1 transmembrane and intracellular domains as well as Hes1 and survivin, well-established downstream targets of Notch1. At clinically achievable concentrations, nelfinavir rapidly inhibited Notch1, which was sustained to 16 hr when death was evident. Cell-free assays showed that although compound E was a more potent inhibitor of γ-secretase, the maximal enzymatic inhibition by nelfinavir was equivalent at higher doses. Nelfinavir was far more effective than compound E in reducing expression of Notch targets such as phospho-Rb, c-Myc, and Hrb, Hes1, and survivin. Nelfinavir induced apoptosis to levels equivalent to 20 µM etoposide, but compound E was only cytostatic. To explore other mechanisms that could explain the decreased protein expression and higher levels of cell death with nelfinavir, we assessed the Akt/mTOR pathway. Nelfinavir rapidly induced AMPK activation and mTOR inhibition, coincident with Notch1 inhibition. In SCL-LMO1 double transgenic mice, nelfinavir decreased thymic size and markedly increased apoptotic cells in the thymus. It also decreased the CD3+ populations in spleen and bone marrow and decreased CD4+CD8+ double positive leukemic cells. Conclusion: These studies show that nelfinavir is superior to a GSI and highly active in preclinical studies of T-ALL. Clinical testing of nelfinavir in T-ALL patients is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2603. doi:10.1158/1538-7445.AM2011-2603

Transcription factor Fli-1 is involved in regulation of dendritic cell and monocyte development (153.33)

Abstract The Fli1 gene is a member of the ets family of transcription factors that is expressed in hematopoietic cells. We have reported that Fli-1 plays an important role in the B cell and megakaryocyte development. C57BL/6 (B6) mutant Fli-1 mice (Fli-1ΔCTA) express a truncated Fli-1 protein that lack the C-terminal transactivation domain Fli-1ΔCTA B6 mice had significant decreased follicular B cells, increased marginal zone B cells and decreased platelets compared with wild-type mice. In this report, we examined the role of Fli-1 in dendritic cell and monocyte development using Fli-1ΔCTA B6 mice. Fli-1ΔCTA and wild-type B6 mice were sacrificed at the ages of 8-12 weeks and dendrtic cell and monocyte populations in blood, spleen and bone marrow were analyzed with Flow cytometery using specific antibodies. Fli-1ΔCTA B6 mice had significantly increased conventional dendritic cells and monocytes in blood compared with wild-type control B6 mice. Also dendritic cell population in the spleen and the common dendtitic cell precursor population in bone marrow are significantly increased compared to wild-type controls. Our results demonstrate that the Fli1 plays an important role in myeloid cell development and the CTA domain in Fli-1 protein is negatively regulates the myeloid cell development. Further molecular mechanisms regarding effects of Fli-1 on myeloid cell differentiation are under investigation.

The role of deltex1 in regulating T cell tolerance (63.20)

Abstract Deltex1 (DTX1) is an E3 ubiquitin ligase and is suggested to be a mediator of Notch signaling. Our previous study found that DTX1 supressses T cell activation and promotes MEKK1 degradation. In this study, we found that T cell activation was partially suppressed by DTX1 with either inactive mutation of RING finger domain or deletion of WWE domain. In addition, we identified another suppressive mechanism of DTX1 by upregulation of Gadd45β, an inhibitor of JNK activation. DTX1 may promote the expression of Gadd45β in the absence of RF domain or WWE domain. Therefore, DTX1 inhibit T cell activation by either E3-dependent or E3-independent mechanisms. To further investigate the physiological function of DTX1 in lymphocyte development and activation, Dtx1-deficient mice were generated. T cell development in thymus and spleen was normal. B cell development in bone marrow was not affected but margine zone B cell population in spleen was reduced in Dtx1-deficient mice. T cell proliferation, cytokine production and MAPK activation were enhanced in Dtx1-deficient T cells. In addition, enhanced autoantibody production, elevated immune complex deposition in kidney and increased leukocyte infiltration in lung and liver were observed in aged Dtx1-deficient mice. These results suggest that DTX1 plays a role in T cell tolerance induction.

CK12, a rainbow trout chemokine with lymphocyte chemo-attractant capacity associated to mucosal tissues

Although many chemokine genes have been identified in rainbow trout (Oncorhynchus mykiss) as in other teleost species, almost no studies focused on their biological role have been conducted, despite the fact that no clear inferences as to their functions can be made based on their low similarity to mammalian counterparts. In the current work, we have studied the regulation of mRNA transcription and protein expression of CK12, a rainbow trout CC chemokine previously catalogued within the CCL19/21/25 phylogenetic group. Our studies revealed that CK12 is strongly expressed both at mRNA and protein level in mucosal tissues. Mature lymphocyte populations also express CK12 both at mRNA and protein levels. Concerning its biological activity, a significant chemotatic activity towards purified recombinant CK12 in unfractionated leukocyte populations was observed in the spleen, but not in head kidney or blood. Consequently, a binding assay revealed that the number of leukocytes capable of binding CK12 was much more elevated in spleen populations than in leukocyte populations from other organs. This binding capacity was only observed in small lymphocytes that should account for resident inactivated lymphocytes, in contrast to mature lymphocytes that were responsible for CK12 production. Around 36% of these small lymphocytes were IgM+ cells, of which 40% had a CK12 binding capacity. On the other hand, 10% of thymocytes were also capable of CK12 binding, suggesting that both T and B immature lymphocytes are recruited by CK12. This work constitutes the first description of a mucosal-associated chemokine in fish in which important aspects of its regulation and functionality are revealed.

Heterotrimeric Gα i proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis

Previous studies have implicated a role of heterotrimeric Gα i proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα i proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα i–GTP protein complex was immunoprecipitated with a Gα i protein activation assay. In subsequent in vivo studies, the Gα i protein inhibitor pertussis toxin (PTx) or G i protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα i2 in sepsis, Gα i2 (−/−) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα i proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα i proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα i2 (−/−) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα i2 (−/−) compared to WT mice. Gα i2 (−/−) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα i proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα i2 protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.

Negative Autoregulation by Fas Stabilizes the Erythroid Progenitor Pool and Accelerates the Erythropoietic Stress Response

AbstractAbstract 2045Signaling and transcriptional networks frequently contain negative autoregulatory feedback loops, where gene products negatively regulate their own induction or activation. These negative autoregulatory motifs are predicted to exert dual functions, accelerating gene induction, and providing stable gene expression levels in the face of the random perturbations inherent to biological systems. These predictions were confirmed experimentally in synthetic transcriptional circuits [1,2], but it is unknown whether they also hold in naturally occurring higher level biological networks. Here we studied the role of negative autoregulation by erythroid progenitors in the control of erythropoiesis.Erythropoietic rate, which may increase ten fold its basal rate during hypoxic stress, is dependent on the size of the erythroid progenitor pool, in turn regulated by the hormone erythropoietin (Epo). We recently found that, in addition, early erythroblasts negatively regulate their own numbers, through their co-expression of the death receptor Fas and its ligand FasL. Here we investigated the role of this negative autoregulation using Fas or FasL-deficient mice. We used the naturally-occuring mutant mouse strains, lpr and gld, deficient in Fas and FasL, respectively, back crossed onto the Rag1-/- mutant background, in order to avoid the autoimmune syndrome associated with Fas mutation. We proceeded to examine basal and stress erythropoiesis in the gld-Rag1-/- and lpr-Rag1-/- mice, and in matched Rag1-/- controls.We found that, in the basal steady state, the average size of the spleen early erythroid progenitor pool in gld-Rag1-/- and lpr-Rag1-/- mice increased 1.5 to 2 fold, consistent with loss of a negative regulator. Further, gld-Rag1-/- mice had a significantly elevated hematocrit in spite of normal Epo blood levels. The hematocrit was normal in the lpr-Rag1-/- mice, but Epo levels in this strain were significantly lower than normal. Taken together, these genetic mouse models show that Fas-mediated apoptosis of early erythroblasts in spleen negatively regulates erythropoietic rate in the basal state.We also found that the size of the progenitor pool was highly variable between individual Fas-deficient mice, suggesting reduced ability to maintain a stable steady-state erythorpoietic rate. In addition, gld-Rag1-/- and lpr-Rag1-/- mice had a significantly delayed erythropoietic stress response. Following an injection of a single dose of Epo (300 U/25 g), the early erythroblast population in spleen, ‘EryA’ (Ter119highCD71highFSChigh, [3]) expanded 30 to 60 fold its basal size. However, this expansion was significantly delayed in gld-Rag1-/- and lpr-Rag1-/- mice. Specifically, on day 2 of the stress response, control Rag1-/- mice had a 30% larger EryA progenitor pool compared with lpr-Rag1-/- mice, a difference equivalent to 10 fold the size of the basal EryA pool. Consequently, control mice achieved a higher hematocrit 24 hours earlier than mutant gld-Rag1-/- and lpr-Rag1-/- mice.We propose that the larger expansion of EryA cells during the stress response in control mice is due to the recruitment of a reserve population of Fas-positive EryA. This reserve population, absent in mice deficient in the Fas pathway, undergoes Fas-mediated apoptosis in the basal steady state. However, high Epo levels during the stress response suppress Fas expression [3], rescuing these cells from apoptosis and accelerating the stress response.These findings show, using genetic mouse models, that the stability of stead-state erythropoietic rate and its rapid stress response are key outcomes of negative autoregulation within the erythroid progenitor pool. Furthermore, they show experimentally that dynamic properties of negative autoregulatory loops in simple low-level networks are also exerted in the context of complex inter-cellular, tissue level networks such as those that regulate erythroipoietic rate.

Bioluminescent Monitoring of Microenvironmental Effects on Multiple Myeloma Engraftment In a Humanized Mouse Model

AbstractAbstract 1927Multiple myeloma (MM) is characterized by clonal proliferation of CD138+ plasma cells in the bone marrow (BM) and remains an incurable disease. Recent identification of a rare population of MM cancer stem cells (MM CSC) is phenotypically similar to memory B cells (CD138- CD34- CD19+) but differs in that they have the self-renewal capacity within the BM and may be responsible for drug resistance. Preclinical testing of novel therapeutic strategies that target MM CSC requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. We have previously reported results on establishing a MM animal model by transplanting MM CSC from autologous mobilized peripheral blood of primary MM patients, transduced with lentiviral luciferase GFP (GLF) and transplanted intrahepatically (IH) into neonatal RAG2/gc double knock-out (RG-KO). Here we evaluate engraftment efficiency in consideration of BM microenvironment by comparing CD45+ human cell engraftment in mice transplanted either IH to neonates or intrafemorally (IF) to gamma-irradiated young adult mice. MM CSC were selected from isolated PBMC after Ficoll gradient centrifugation of fresh BM biopsy from two primary MM patients or from a human MM cell line, H929, followed by immunomagnetic bead depletion of CD34+ and CD138+ cells. The cells were transplanted into RG-KO mice ranging from 53,000 to 10⋀6 cells per mouse either IH or IF. Mice transplanted with GLF-transduced MM CSC were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that bioluminescence signal levels were detected in mice transplanted IF with 53,000 MM CSC per mouse even before 3 weeks by ventral view and as early as 5 weeks by lateral view. To date, tumor growth was only discovered in mice transplanted IH with 2 × 10⋀6 unselected MM PBMC from a fresh BM biopsy as early as 10 week post-transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+, CD19+ and CD138+ population in tumor, bone marrow, spleen and liver. In addition, expression of clonal light chain restriction in myeloma cells confirmed myeloma engraftment. Future studies will focus on expression of genes involved in sonic hedgehog pathway as analyzed by PCR to confirm the self-renewal capacity. Moreover, investigations on the effect of B cell-activating factor (BAFF) in BM microenvironment by transplanting MM CSC into BAFFxRG-KO mice are in progress. Disclosures:Jamieson:Bristol-Meyers Squibb: Research Funding.

Adoptively Transferred Mature T Cells Exhibit Diminished Reactivity to Murine Cytomegalovirus Compared to Nascent Donor T Cells and Impair Functional Immune Recovery After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 3732Infections due to impaired immune function are a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Adoptively transferred donor (do) T cells (TC) are thought to provide protective immunity, as TC-depleted grafts are associated with increased viral infections post HCT (pTX). TC-replete grafts, however, require immunosuppression to prevent GVHD, and can damage lymphoid organs causing additional immune dysfunction. Using an MHC-matched, minor antigen (miAg) mismatched GVHD model, we compared immune responses to murine cytomegalovirus (MCMV) in lethally irradiated BALB.B recipients infused with purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin−) or HSC+TC (subsets) from congenic C57BL/6 (B6) donors which could be distinguished based upon CD45 alleles. Hosts were infected with sublethal doses of MCMV at 2 or 8 weeks (w) pTX. 2w after infection, lymphoid organs were harvested to assess the immune function of: 1) transferred doTC from uninfected and pre-immunized donors, 2) HSC-derived doTC, and 3) residual host TC. Anti-MCMV CD8 TC were measured by M45 tetramer staining and an MCMV ELISPOT assay was used to measure IFNg production of FACS-separated doTC-, doHSC-, and host-derived spleen populations when exposed to the viral peptide. Mice given TC-replete allografts and infected at 2w pTX mounted significantly lower responses than control congenic- or HSC only recipients. In the latter group reactivity originated from residual host cells. Transplantation of TC from immunized donors did not improve the response of doTC transplanted into miAg-mismatched hosts. However, when HSC were supplemented with memory (CD62L+/−CD44+) CD8 TC from immunized donors, recipients of memory, but not of naive CD8 TC (CD62L+CD44−), mounted strong anti-MCMV responses in both assays. At 8w pTX recipients of pure HSC were mixed TC chimeras, while mice given HSC+TC were full donor chimeras with the majority of TC originating from transferred doTC. When mice were infected 8w pTX, anti-MCMV reactivity of CD8 TC was significantly greater in recipients of pure HSC, as compared to weak responses seen in recipients of HSC+TC. In recipients of pure HSC, both donor and host populations demonstrated strong reactivity, while mice given HSC+TC had weak responses of both doTC-, and doHSC-derived TC (Fig.1). When recipients of HSC alone or HSC + total TC (ToTC), naive CD8, memory CD8 or M45 tetramer+ CD8 TC were infected with a lethal dose of MCMV at 2w pTX, 50% of ToTC and 20% of HSC recipients died before day 40 pTX, while all mice given any of the CD8 subsets were protected from infection. 6w post MCMV infection organs of survivors were analyzed. Hepatic CD8 cells contained a median of <0.05% M45 tetramer reactive cells in ToTC-, <0.5% in HSC only- and naive CD8-, and 0.65% in memory CD8 recipients (p=0.03 compared to ToTC group). Strikingly, in mice given as few as 7,000 M45 tetramer+ cells, the proportion of M45-reactive cells reached a median 8.7% in the liver, and 4.3% in the spleen. In mice given ToTC, >90% of TC were doTC-derived with an effector memory phenotype. In contrast, recipients of naive, memory, or tetramer-sorted CD8 cells had TC originating from expanded doTC, doHSC, or residual host. Nascent doHSC-derived lymphopoiesis sustained a robust pool of naive TC - a sign of healthy immune reconstitution. Thus, our key findings are: 1) mature doTC do not maintain their full anti-viral potential when transplanted into miAg-disparate mice, even when obtained from pre-immunized donors; 2) residual host cells contribute substantially to protective immunity early pTX, but are eradicated by conventional TC-replete grafts; 3) HSC-derived TC arising in a healthy host are superior to those that develop and undergo selection in a GVHD-affected lymphoid system; and 4) low numbers of selected cell subsets, such as tetramer-sorted CMV-specific CD8 TC, expand dramatically in a lymphopenic environment and provide functional protection against infections. Our results challenge the conventional assumption that doTC in a hematopoietic allograft are required for optimal regeneration of the immune system. Rather, our studies suggest that long-term lymphoid function will greatly benefit from rigorous TC-depletion of the graft, and avoidance of GVHD. Moreover, co-transfer of small numbers of highly selected TC clones can provide effective antigen-specific protection. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The proteasome inhibitor bortezomib drastically affects inflammation and bone disease in adjuvant‐induced arthritis in rats

To explore the effect of bortezomib in splenocytes and fibroblast-like synoviocytes (FLS) and its in vivo potency in a rat model of adjuvant-induced arthritis (AIA), which resembles human rheumatoid arthritis (RA). AIA was induced with Freund's complete adjuvant. Splenocyte and FLS proliferation and apoptosis were measured by radioactivity incorporation and flow cytometry, respectively. The invasiveness of FLS from rats with AIA was tested in a Transwell system. The pattern of cytokine secretion was evaluated by cytometric bead array in splenocyte supernatants. Bortezomib was administered prophylactically or therapeutically, and arthritis was assessed clinically and histologically. Immunohistochemistry was performed for markers of inflammation and angiogenesis in joints. Hematologic and biochemical parameters were tested in peripheral blood (PB). Representative animals were examined by computed tomography (CT) scanning before and after bortezomib administration. The expression of Toll-like receptor 2 (TLR-2), TLR-3, and TLR-4 in PB and FLS was measured by real-time polymerase chain reaction, and alterations in specific cell populations in PB and spleen were determined by flow cytometry. In vitro, bortezomib exhibited significant inhibitory and proapoptotic activity in splenocytes and FLS from rats with AIA, altered the inflammatory cytokine pattern, and reduced the invasiveness of FLS from rats with AIA. In vivo, bortezomib significantly ameliorated disease severity. Remission was associated with improved histology and decreased expression of CD3, CD79a, CD11b, cyclooxygenase 1, and factor VIII in target tissues as well as down-regulation of TLR expression in PB and cultured FLS. CT scanning demonstrated a bone healing effect after treatment. Our findings suggest that bortezomib affects AIA in a pleiotropic manner and that this drug may be effective in RA.