Addition Of IL-2 Research Articles

A Functional IL-13 Receptor Is Expressed on Polarized Murine CD4+ Th17 Cells and IL-13 Signaling Attenuates Th17 Cytokine Production

IL-17A is produced from Th17 cells, and is involved in many autoimmune and inflammatory diseases. IL-13R has not previously been reported to be functionally expressed on T cells; however, we found that purified BALB/c CD4(+) cells polarized to Th17 with TGF-beta, IL-6, and IL-23 have increased mRNA and protein expression of IL-13R alpha1 and mRNA expression of IL-4R alpha compared with Th0, Th1, or Th2 polarized cells. The addition of IL-13 at Th17 polarization negatively regulated IL-17A and IL-21 expression, and reduced the number of CD4(+) T cells producing IL-17A. Further, adding IL-13 at the time of Th17 cell restimulation attenuated IL-17A expression. CD4(+) Th17 polarized cells from IL-4 knockout (KO) mice also had IL-13-induced inhibition of IL-17A production, but this was not observed in IL-4R KO and STAT6 KO mice. Addition of IL-13 at polarization increased IL-13R expression in wild-type Th17 cells. Further, IL-13 administration during Th17 polarization down-regulated retinoic acid-related-gammaT, the transcription required for Th17 development; increased STAT6 phosphorylation, and up-regulated GATA3, the transcription factor activated during the development of Th2 cells. This IL-13-mediated effect was specific to Th17 cells as IL-13 neither decreased IFN-gamma expression by Th1 cells nor affected Th2 cell production of IL-4. Collectively, we have shown that Th17 cells express a functional IL-13R and that IL-13 negatively regulates IL-17A and IL-21 production by decreasing retinoic acid-related-gammaT expression and while increasing phosphorylation of STAT6 and GATA3 expression. Therefore, therapeutic intervention inhibiting IL-13 production could have adverse consequences by up-regulating Th17 inflammation in certain disease states.

Glucose Metabolism Attenuates p53 and Puma-dependent Cell Death upon Growth Factor Deprivation

Growth factor stimulation and oncogenic transformation lead to increased glucose metabolism that may provide resistance to cell death. We have previously demonstrated that elevated glucose metabolism characteristic of stimulated or cancerous cells can stabilize the anti-apoptotic Bcl-2 family protein Mcl-1 through inhibition of GSK-3. Here we show that the pro-apoptotic Bcl-2 family protein, Puma, is also metabolically regulated. Growth factor deprivation led to the loss of glucose uptake and induction of Puma. Maintenance of glucose uptake after growth factor withdrawal by expression of the glucose transporter, Glut1, however, suppressed Puma up-regulation and attenuated growth factor withdrawal-induced activation of Bax, DNA fragmentation, and cell death. Conversely, glucose deprivation led to Puma induction even in the presence of growth factor. This regulation of Puma expression was a central component in cell death as a consequence of growth factor or glucose deprivation because Puma deficiency suppressed both of these cell death pathways. Puma induction in growth factor or glucose withdrawal was dependent on p53 in cell lines and in activated primary T lymphocytes because p53 deficiency suppressed Puma induction and delayed Bax and caspase activation, DNA fragmentation, and loss of clonogenic survival. Importantly, although p53 levels did not change or were slightly reduced, p53 activity was suppressed by elevated glucose metabolism to inhibit Puma induction after growth factor withdrawal. These data show that p53 is metabolically regulated and that glucose metabolism initiates a signaling mechanism to inhibit p53 activation and suppress Puma induction, thus promoting an anti-apoptotic balance to Bcl-2 family protein expression that supports cell survival.

Lineage-Specific Regulation of miRNA-Expression by the BCR-ABL Oncogene.

Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′-untranslated region of corresponding mRNAs resulting in inhibition of mRNA translation or mRNA degradation. Aberrant expression of specific miRNAs has been described in a variety of human malignancies but its functional consequences are currently largely unknown. The miR-17- 92 cluster (encoding miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been shown to be over-expressed in chronic phase but not in blast crisis CML CD34+ cells in a BCR-ABL- and c-Myc-dependent manner (Venturini et al. 2007). Interestingly, the knock-out of the polcystronic miR-17-92 cluster in mice recently revealed an essential role in lung development and B-lymphopoiesis at the stage of pre-B-cells also affected in pre- B-ALL which often expresses the BCR-ABL oncogene. To study miR-17-92 expression in BCR-ABL + myeloid and lymphoid cells we performed miRNA specific quantitative RT-PCR (miR-qRT-PCR) in myeloid human K562 and murine 32D/BCR-ABL cells and in lymphoid human BV173 and IL-3-dependent murine TonB cells which can be induced to express BCR-ABL. We found reduced expression of both the miR-17-92 cluster and c-MYC in lymphoid as compared to myeloid cells. Inhibition of BCR-ABL tyrosine kinase activity by imatinib reveals that miR-17-92 expression depends on BCR-ABL in myeloid K562 and 32D/BCR-ABL but not in lymphoid BV173 and TonB cells. Similarly, RNAi against c-MYC reduces miR-17-92 expression in the myeloid but not in lymphoid cell lines demonstrating lineage-specific effects on BCR-ABL-mediated regulation of miR17-92 expression. Furthermore, preliminary chromatin immunoprecipitation analyses in the presence and absence of imatinib demonstrate BCR-ABL tyrosine kinase dependent recruitment of c-MYC to the miR-17-92 5′-regulatory region in myeloid, but not in lymphoid cells. Correspondingly, transcription of the miR-17-92 pri-miRNA is reduced by imatinib treatment in myeloid cell lines. To study the function of individual miRNAs encoded within the polycistronic miR-17-92 cluster in the presence and absence of BCR-ABL, TonB cells were lentivirally transduced with miR-17-19b, a variant of miR- 17-92 selected for efficient transgenic miRNA expression, and with individual miRNAs embedded within miR-30-derived sequences to induce stable miRNA-specific gain-offunction phenotypes (Scherr et al. 2007). Transduction rates were about 98%, and miRqRT- PCR demonstrated increased miRNA-expression between 2- and 14-fold for miR-17- 5p, miR-19b, and miR-20a, respectively. Over-expression of these three miRNAs and the miR-17-19b polycistron had no effect on cell proliferation of TonB cells under stimulation with IL-3 as compared to controls. In contrast, BCR-ABL-dependent proliferation in the absence of IL-3 was reduced by miR-17-5p by about 80% and completely by miR-17-19b in two experiments, whereas miR-19b and miR-20a had only minor inhibitory effects. Interestingly, addition of IL-3 to BCR-ABL+ TonB cells can rescue cell proliferation for all these miRNAs. These data demonstratelineage-specific effects of BCR-ABL on expression of the miR-17-92 cluster andmay suggest that miRNAs encoded within this polycistron encompass target genes whose expression is required for BCR-ABL-mediated cell transformation.

Cellular Tests in the Diagnosis of Drug Hypersensitivity

The application of flowcytometry in the study of basophil activation for the diagnosis of allergic diseases has given interesting results in recent years. The quantification of basophil activation by flowcytometry has been proven to be a useful tool for the assessment of the immediate-type response to allergens mediated by IgE or by other mechanisms in drug allergic patients. Up to now, most basophil activation test studies reported in the literature have used CD69 or CD203c as markers to quantify basophil activation after antigen-specific stimulation. Some technical variations such as the use of whole blood or isolated leukocytes, the addition of IL-3, the conditions of storage of the blood sample, the time of incubation with allergens and their concentration can affect the results of the basophil activation tests. The basophil activation test is more sensitive and specific than other in vitro diagnostic techniques in drug allergy. In various studies, its sensitivity in allergy to muscle relaxant drugs ranges between 36 and 97.7%, with a specificity around 95%. For betalactam antibiotics, basophil activation test sensitivity is 50% and its specificity 90%. For NSAIDs, sensitivity varies between 66% and 75%; specificity is about 93%. Basophil activation test reproduces in vitro hypersensitivity mechanisms involved in immediate-type allergic reactions, allows the diagnosis of allergic and pseudo-allergic reactions particularly for drugs, which are often not detectable by serological techniques, such as determination of specific IgE.

Differential cytokine expression and regulation of human anti‐pig xenogeneic responses by modified porcine dendritic cells

Porcine dendritic cells (DC) are likely to be pivotal cells in the initiation of stimulatory and potential tolerogenic responses to xenoantigens, however, there are limited studies characterizing these antigen presenting cells. Porcine PBMC (CD172a(+)) were cultured with GM-CSF and IL-4 and phenotype and functional capabilities assessed. Lipopolysaccharide (LPS), IL-10, and IL-3 were added to the GM-CSF/IL-4 DC cultures to determine phenotypic and functional changes. Quantitative real-time polymerase chain reaction (PCR) for key cytokines was performed and the modified porcine DC were further assessed by primary mixed lymphocyte reaction to determine the effect of LPS, IL-10, and IL-3 on stimulatory capability. Porcine PBMC (CD172(+)) cultured with GM-CSF and IL-4 produced cells with DC morphology, which were major histocompatability complex (MHC) class II(+), CD14(-/lo), and CD1a(lo). Addition of IL-10 or IL-3 to GM-CSF/IL-4 DC cultures produced cells with lower levels of MHC class II and higher levels of antigen uptake consistent with less mature DC. Quantitative real-time PCR of DC showed the addition of IL-10 induced an increase in IL-10 mRNA, no detectable IL-12, and reduced IL-6 mRNA. The addition of IL-3 to DC cultures decreased IL-12, IL-6 and tumor necrosis factor (TNF), with no change in IL-10 mRNA. GM-CSF/IL-4 DC induced strong human lymphocyte proliferation, compared with significantly reduced stimulatory capacity induced by IL-10 and IL-3 treated DC cultures. The profound effect on differential DC cytokine profile and reduced human anti-pig responses has important therapeutic implications in xenotransplantation. The mechanism of altered regulation warrants further investigation.

The effect of cytokines on the in-vitro erythropoietin bioassay.

The effects of various concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (gamma IFN) and interleukins 1 alpha (IL-1 alpha), 1 beta (IL-1 beta) and 3 (IL-3) on the anaemic mouse spleen cell bioassay for erythropoietin (EPO) were investigated. Addition of IL-3 and GM-CSF at various concentrations had no effect on EPO stimulated 3H thymidine incorporation. However the addition of IL-1 alpha, IL1-beta and gamma IFN (3.3 x 10(-8) gl-1) caused a significant (P < 0.01) inhibition of EPO stimulated thymidine incorporation. This suggests that the EPO bioassay may be influenced by variable levels of some inflammatory cytokines in serum. Previous studies have shown that the bioassay is influenced by serum transferrin levels and thus serum immunoassays remain the technique of choice for specific estimates of EPO. Since EPO bioassays are not specific, they should be reserved for situations in which an estimate of the total erythropoietic activity of serum is required.

Comparison of short term in vitro cultured human mast cells from different progenitors — Peripheral blood-derived progenitors generate highly mature and functional mast cells

During the last two decades different scientific groups have investigated the phenotype and function of in vitro generated human mast cells (MC). The cells have been shown to display variable surface markers and functional characteristics. The phenotypic differences may reflect different culture conditions, protocols or the use of different progenitors.To investigate the significance of different progenitors, we have compared MC generated from CD133+ progenitor cells from cord blood (CBMC) or peripheral blood (PBMC). The progenitors were cultured for 7 weeks in the presence of IL-6 and SCF, with addition of IL-3 the first 3 weeks, and FCS during week 7. The phenotype of the established MC was characterized by surface marker expression levels, metachromasia, histamine and tryptase contents and their function was evaluated by receptor-mediated release of histamine and PGD2.The generated metachromatic (<99%) MC were 75% tryptase+, regardless of the source of progenitor cell. Expression of c-kit/CD117, CD203c, and FcεRI was comparable. The density of c-kit/CD117 receptors on CBMC was higher that of PBMC (p<0.001). The density of CD203c and FcεRI was higher on PBMC (p<0.001). PBMC contained more histamine (p<0.001), expressed more FcεRI (p<0.001) and released more histamine (p<0.001) and PGD2 (p<0.001) upon ligation of FcεRI, than CBMC.Culture with IL-4 increased expression of tryptase, FcεRI, CD117 and CD203c, secretion of histamine and PGD2 of PBMC, and histamine secretion of CBMC.Cord and peripheral blood may give rise to different types of MC. The question addressed should determine the progenitor cell and protocol to be used.

Mouse fetal and embryonic liver cells differentiate human umbilical cord blood progenitors into CD56-negative natural killer cell precursors in the absence of interleukin-15

Human natural killer (NK) cell maturation involves the orderly acquisition of NK cell receptors. Our aim was to understand how stromal interactions and cytokines are important in this developmental process. Human umbilical cord blood (UCB) CD34(+)/Lin(-)/CD38(-) cells were cultured on two murine stromal cell lines (AFT024 and EL08-1D2) in a switch culture to study NK cell development. When human progenitors were cultured on AFT024 with interleukin (IL)-3 and Flt3 ligand (Flt3L) in the absence of interleukin (IL)-15, NK cell differentiation occurred, albeit at low frequency. These conditions favored the accumulation of CD56(-) NK cell precursors (CD34(+)CD7(-), CD34(+)CD7(+), and CD34(-)CD7(+) cells), which are populations rare in adult blood but abundant in fresh UCB. In secondary culture, addition of IL-3 or IL-3 + Flt3L to IL-15 increased the absolute number of CD56(+) NK cells from precursors and the acquisition of CD94 and killer immunoglobulin-like receptors (KIR). To further explore the microenvironment in early NK cell maturation, a cell line derived from murine embryonic liver (EL08-1D2) was studied. NK cell development and KIR acquisition was superior with EL08-1D2, which supported the differentiation of NK cell precursors, NK cell commitment, and proliferation. Although the earliest events in NK cell maturation do not require exogenous human IL-15, it is required at a later stage of NK cell commitment. At a minimum, murine stroma, IL-3, and Flt3L are required to recapitulate early NK cell development and differentiation into distinct NK cell precursors. EL08-1D2 induces KIR acquisition suggesting that extrinsic signals in NK cell development are conserved between mouse and man.

Discovery and Preclinical Characterization of INCB018424, a Selective JAK2 Inhibitor for the Treatment of Myeloproliferative Disorders.

Activating mutations in Janus kinase 2 (JAK2) have recently been identified in the majority of Philadelphia chromosome negative (Ph-) myeloproliferative disorders (MPDs). Importantly, constitutive JAK2 activation is oncogenic and, in murine models, recapitulates much of the pathobiology observed in MPD patients, suggesting that JAK2 inhibition may be of therapeutic benefit. Here we describe the identification and preclinical characterization of INCB018424, a potent, selective, and orally bioavailable inhibitor of the JAK2 now in clinical trials. INCB018424 was identified through an extensive medicinal chemistry effort designed to optimize potency, selectivity, pharmaceutical and pharmacokinetic properties. INCB018424 inhibits JAK2 at <1 nM and demonstrated >500–fold selectivity against a broad sampling of the kinome. The potency and selectivity of INCB018424 translated to exceptional cellular activity where it inhibited the proliferation of FDCP cells and BaF/3 cells expressing JAK2V617F with an IC50 of 100–130 nM, but not the proliferation of cell lines expressing activating mutations in either BCR-Abl or cKit (IC50 > 25 and 4 mM, respectively). The effect of INCB18424 on cell proliferation correlated well with reduced levels of phosphorylated JAK2 and STAT5 in the BaF/3 cell model, suggesting that the effect is mediated by pharmacological inhibition of JAK-STAT pathway. Interestingly, the activation of endogenous wild-type JAKs - by the addition of IL-3 - shifted the potency of INCB018424 in the BaF/3 model greater than five fold suggesting that cells expressing the mutated form of JAK2 may be more sensitive to INCB018424. Indeed, using cells harvested from patients with Jak2V617F-positive polycythemia vera (PV) in colony forming assays, we observed that INCB018424 inhibited the cytokine-independent formation of erythroid progenitor colonies (n=3) with an IC50 of 67nM while normal colony formation from healthy donors (n=3) was inhibited 50% at > 400 nM. Further, INCB018424 inhibited proliferation of PV patient samples (n=3) following ex vivo expansion of erythroid progenitors in serum free media, with an IC50 of 60 nM, similar to that observed in semi-solid media. In a mouse model of MPD, where implantation of BaF/3 cells expressing JAK2V617F results in rapid organomegaly and reduced survival, oral administration of INCB018424 was well tolerated and markedly reduced the splenomegaly. Using this animal model, we also demonstrated that selective JAK inhibition eliminates neoplastic cells from the spleen, liver, and bone marrow normalizing the histology of affected organs and significantly prolongs survival. As such, potent and selective JAK inhibitors such as INCB018424 hold great promise for the treatment of MPDs and other disease states associated with elevated JAK activity - a concept currently being tested clinically.

Gene Transfer of IL-7R alpha (IL-7Rα) on Antigen-Specific Cytotoxic T Cells (CTLs) Restores Their Ability To Respond to IL-7 Cytokine.

Systemic administration of IL-2 may improve the expansion and persistence of adoptively transferred anti-tumor CTLs. However, significant toxicity and concomitant expansion of regulatory T cells (Tregs) limit the clinical value of this strategy. IL-7 plays a crucial role in maintaining T-cell homeostasis, and administration appears well tolerated and not to expand Tregs. Nonetheless, the value of IL-7 for promoting proliferation of tumor-specific CTLs after adoptive transfer remains questionable, due to the lack of expression of the IL-7 receptor (IL-7Rα) on effector CTLs. Thus we have found IL-7Rα to be essentially undetectable in our model of EBV-specific CTL lines (EBV-CTLs) due to downregulation of IL-7Rα transcripts. To determine whether transgenic expression of IL-7Rα can restore the capacity of established EBV-CTLs to respond to IL-7, the full-length of IL-7Rα was cloned in the SFG retroviral vector (SFG/IL-7Rα). We transduced EBV-CTLs generated from 5 healthy donors and evaluated their growth kinetics and antigen specificity. As control, EBV-CTLs were transduced with the SFG retroviral vector encoding for a truncated form of the CD34 molecule (SFG.ΔCD34). After transduction with SFG/IL-7Rα, IL-7Rα was detectable in 58% to 76% of EBV-CTLs. The transgenic IL-7Rα was functional since addition of IL-7 (2ng/mL) to transgenic cells induced phosphorylation of STAT5 within 10 min. To evaluate whether rhIL-7 sustained the expansion of EBV-CTLs/IL-7Rα+, transduced CTLs were stimulated weekly with autologous EBV-LCLs in the presence of IL-2 (50U/mL) or IL-7 (2ng/mL). Control EBV-CTLs and EBV-CTLs/IL-7Rα+ expanded equally well with IL-2. However, only EBV-CTL/IL-7Rα+ significantly proliferated in the presence of IL-7 [from 1×106 cells to 103×106 cells (range, 38-286×106)] over a period of five weeks. CTL expansion remained antigen dependent, and antigen withdrawal led to cessation of CTL growth. EBV-CTLs/IL-7Rα+ showed the expected growth advantage in the presence of IL-7, increasing from 55%±15% to 79%±5% of the total within 5 weeks. In contrast, the proportion of IL-7Rα+ cells marginally declined when the cells were expanded with IL-2 (from 64%±14% to 40%±11%). Importantly, EBV-CTLs/IL7Rα+ expanded with IL-7 retained their ability to respond to other common-γ-chain cytokines. EBV-CTLs/IL7Rα+ grown with IL-7 for more than 2 weeks, expanded further when exposed to both antigen and IL-2 or IL-15. Control CTLs grown with IL-2 and EBV-CTLs/IL-7Rα+ grown with IL-7 both remained polyclonal, as assessed by VβTCR repertoire analysis, and were mostly CD3+/CD8+ (> 90±8%) with an effector-memory profile. EBV-CTLs/IL-7Rα+ also retained antigen specificity measured by tetramer staining, by IFNγ release in response to EBV-peptides and by MHC-restricted killing of autologous LCLs (52%±18% at a ratio 20:1 vs 9±17% of allogeneic LCL); this killing was comparable to the activity of control CTLs cultured in IL-2 (51%±27%). Preliminary studies indicate that EBV-CTLs/IL-7Rα+ also expand in vivo in response to IL-7 and maintain their anti-tumor activity in a SCID mouse xenograft model.

Activating PP2A Tumor Suppressor Activity as a Potential Therapy for Treating Jak2 V617F-Driven Myeloproliferative Disorders.

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) caused by a valine to phenylalanine mutation in the inhibitory JH2 “pseudokinase” domain of the signaling protein Janus Kinase 2 (Jak2). While Jak2 has several known regulators (e.g. SOCS1, SOCS3, PP2A, and SHP-1), the role played by these signaling molecules in the development of PV is still largely unclear. One of these regulators, the tumor suppressor phosphatase PP2A, has been found functionally inactivated in different hematologic myeloid and lymphoid malignancies characterized by the expression of constitutively activated oncogenic tyrosine kinases (e.g. BCR/ABL). To investigate the role of PP2A in the pathogenesis of Jak2 V617F+ MPDs and the potential therapeutic relevance of PP2A activating drugs (e.g. FTY720), we determined the effects of wild type and V617F Jak2 expression on PP2A activity and assessed the molecular and biological effects of FTY720 in hematopoietic precursor cell lines and/or primary lineage-negative bone marrow cells engineered to expressed either wild type or V617F Jak2 protein. Herein we report that PP2A activity is significantly reduced by about 82% and 78% (P< 0.01) in mJak2 V617F-transduced growth factor-dependent and erythropoietin receptor-expressing 32Dcl3 (32D-EPO) and Ba/F3 cells, respectively, compared to MigR1-transduced controls. Furthermore, addition of the PP2A activator FTY720 (2.5μM) for 10 hours restored PP2A activity to 66% and 75% respectively compared to that of MigR1 controls. Mechanistically, we demonstrated that inactivation of PP2A was due to constitutive Jak2 activity. In fact, treatment of V617F Jak2-expressing cells with Jak inhibitor I (1μM; 10 hours) restored PP2A activity to 80% of controls in 32Dcl3 cells, while 600nM was sufficient to restore activity to 108% of controls in Ba/f3 cells. Likewise, transduction of murine lineage-negative bone marrow cells with wild-type Jak2 produced a 62% reduction in PP2A activity (P<0.01), while expression of the Jak2 V617F mutant produced a 97% reduction (P< 0.01). Moreover, as we previously reported for BCR/ABL, pharmacologic restoration of PP2A activity by treatment of Ba/F3 or 32D-EPO cells with the PP2A activator FTY720 (2.5μM for 10 hours) led to reduced expression and dephosphorylation of wild type and V617F Jak2. This, in turn, resulted in a 60% reduction in the colony forming ability of IL-3 cultured cells overexpressing wild type Jak2, while a 94% suppression of colony formation was evident in Jak2 V617-expressing cells maintained in the absence of cytokines. Moreover, addition of IL-3 to the FTY720-containing semisolid medium restored viability to levels similar to those of FTY720-treated wild type Jak2-expressing cells (70% suppression of colony formation upon treatment), indicating that restoration of PP2A activity counteracts cytokine-independent pathways triggered by the V617F Jak2 mutated tyrosine kinase. Altogether, these results suggest that inactivation of PP2A is essential for Jak2 (V617F mutant included) -driven cell/proliferation and survival and that pharmacologic activation of PP2A might represent a potential avenue for treatment of PV and, perhaps, of other MPDs characterized by the expression of a mutated Jak2 kinase.

Overexpression of Rheb2 Has Diverse Effects on Hematopoietic Progenitor and Stem Cell Functions.

Determining which intracellular signaling pathways are important for hematopoietic stem and progenitor cell functions is important for therapeutic uses of these cells. The intracellular serine/threonine kinase mammalian target of rapamycin (mTOR) is important for regulation of the growth, proliferation, survival and differentiation of various cell types in the body, including normal hematopoietic and blood cancer cells. We chose to study the protein Ras homologue enriched in brain-2 (Rheb2), a known activator of mTOR signaling, because there is evidence it is preferentially expressed in highly purified mouse (m) hematopoietic stem cell populations (Ivanova et al. Science , 298(5593):601–04, 2002). Rheb2 is one of two members of the Rheb small GTPase family of proteins known to activate mTOR by mechanisms which are not fully elucidated, but that are regulated by Akt and the tuberous sclerosis complex (TSC1/2). We overexpressed mouse Rheb2 protein in both primary mouse bone marrow (mBM) cells and two mouse hematopoietic cell lines (BaF3 and 32D) and determined effects on progenitor cell colony-formation, delayed IL-3 driven proliferation in cell lines and hematopoietic stem cell function in an in vivo competitive stem cell repopulation assay. Overexpression of Rheb2 in retrovirally transduced mBM cells significantly increased the colony-forming ability of the cells in response to growth factors. The positive effect of Rheb2 was most pronounced on granulocyte-macrophage (CFU-GM) progenitors (greater than two-fold increase over empty vector transduced cells) and was persistent in cells expanded in liquid culture up to one week after FACS sorting of transduced cells. Additionally, Rheb2 overexpression significantly increased the ability of mBM cells to form CFU-GM colonies under conditions of delayed-growth factor addition (at 24 and 48 hours after plating), suggesting a positive role for Rheb2 in the survival of these cells. Interestingly, addition of the pro-survival chemokine SDF-1/CXCL12 to the delayed-growth factor colony assay plates enhanced the survival of the empty vector transduced cells but did not further enhance the survival of the Rheb2 overexpressing cells. Using two IL-3 dependent hematopoietic cell lines, BaF3 and 32D, to further assess Rheb2 function and signaling, we found overexpression of Rheb2 enhanced proliferation after delayed addition of IL-3. These changes correlated with an increase in mTOR signaling in Rheb2 transduced cells. In order to evaluate the effect of Rheb2 overexpression on hematopoietic stem cell function, an in vivo competitive repopulation assay was performed. Transduced donor and non-transduced competitor cells were pooled at two ratios (1:1 and 0.4:1 donor to competitor) and injected into lethally irradiated recipients. Transduced cells were followed in the recipients by measuring GFP+ cell chimerism. In contrast to the data on progenitor cell function, transduced Rheb2 cells had significantly decreased competitive repopulation in the recipient mice (greater that 3-fold decrease compared to empty vector). This suggests that Rheb2 overexpression may negatively affect HSC homing, stable engraftment and/or self-renewal. Overall, these studies demonstrate that overexpression of Rheb2 positively enhances progenitor cell functions such as colony-formation, while negatively affecting stem cell competitive repopulation, thereby providing a possible regulatory mechanistic link between the flow from the stem to progenitor cell compartment.

Transcriptional suppression of cytokine-induced iNOS gene expression by IL-13 through IRF-1/ISRE signaling

IL-13 has been reported as one of the major down-regulators of iNOS expression in various tissues and cells. The molecular mechanism of iNOS suppression by IL-13 remains unclear, especially at the transcriptional stage. In this study, we found that IL-13 inhibited the expression of iNOS mRNA, protein, and NO product in a concentration-dependent manner for cytokine-stimulated rat hepatocytes. The most effective dose for IL-13 inhibitory effect is ∼5 ng/ml. IL-13 also decreased the rat iNOS transcriptional activity by promoter analysis, but had no effect on iNOS mRNA stability. By using TranSignal Protein/DNA Combo Array, we identified cytokine-stimulated IRF-1/ISRE binding that was decreased by the addition of IL-13. Gel shift assay confirmed that IL-13 reduced the IRF-1/ISRE binding at nucleotides −913 to −923 of the rat iNOS promoter. Western blot revealed that IL-13 diminished the relative amount of IRF-1 protein translocated to the nucleus. Our data demonstrate that IL-13 down-regulates the cytokine-induced iNOS transcription by decreasing iNOS specific IRF-1/ISRE binding activity.

Profound Enhancement of the IL-12/IL-18 Pathway of IFN-γ Secretion in Human CD8+ Memory T Cell Subsets via IL-15

Human memory CD8(+) T cell subsets, termed central memory and effector memory T cells, can be identified by expression of CD45RA, CD62 ligand (CD62L), and CCR7. Accordingly, functional differences have been described for each subset, reflecting unique roles in immunological memory. The common gamma-chain cytokines IL-15 and IL-7 have been shown to induce proliferation and differentiation of human CD8(+) T cell subsets, as well as increased effector functions (i.e., cytokines, cytotoxicity). In this study, we observed that addition of IL-15 or IL-7 to cultures of human CD8(+) T cells profoundly enhanced the IL-12-IL-18 pathway of IFN-gamma production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction of IFN-gamma by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior in its ability to enhance IL-12-IL-18-induced IFN-gamma, without evidence of a synergistic effect between IL-15 and IL-7. We also observed that IL-15- and IL-7-mediated enhancement of IL-12-IL-18-induced IFN-gamma production was a functional property of effector memory CD8(+) T cells. Despite a lack of association between cell division and acquisition of IL-12-IL-18-induced IFN-gamma, down-regulation of CD62L expression correlated well with increased IL-12-IL-18-induced IFN-gamma. Purified central memory T cells stimulated with IL-15 and IL-7 down-regulated CD62L and acquired potent IL-12-IL-18-induced IFN-gamma similar to effector memory T cells. Thus, in addition to its known role in development of T cell memory, IL-15 may amplify memory CD8(+) T cell effector functions by increasing sensitivity to proinflammatory cytokine stimulation.

Mechanisms of Intestinal Epithelial Cell Regeneration from Radiation-Induced Small Intestinal Injury in Mice

Background: Signaling by IL-4 and IL-13 is transduced via a receptor complex of IL-13Rα1 and IL-4Rα chains. However, contribution of a putative decoy receptor of IL-13, IL-13Rα2, is not clear. The aim of this study was to determine the significance of Th2-type cytokines in the gastrointestinal (GI) epithelial cell repair system. Methods: We compared the tissue recovery in wild type (WT) and IL-4 receptor α deficient (IL-4R KO) mice following mucosal damage induced by 3 Gy-whole body γ-irradiation. The expression of IL-4, IL-13 and related molecules in the regenerative process were assessed. Results: Expression of IL-13Rα2 dramatically increased in myofibroblasts and fibroblasts on day 3. In contrast, in IL-4R KO mice, intestinal tissue repair after irradiation was delayed, and the transient upregulation of IL-13Rα2 was not seen. Injection of a soluble IL-13Rα2-immunoglobulin chimeric protein to both of WT and IL-4R KO mice resulted in the improvement of tissue recovery. Addition of IL-13 into primary tissue culture of the jejunum induced epithelial cell damage with decreased expression of membrane bound β-catenin. Conclusions: Our study demonstrated that IL-13 has a potential to induce epithelial cell damage, and IL-13Rα2 functions as a regulatory factor in the regeneration of intestinal epithelial cells.

PI3-Kinase Inhibition Decreases the Proliferation of ALL Cells, Potentiates mTOR Inhibition, and Abrogates Growth Factor-Mediated Reversal of mTOR Inhibition.

Biologically targeted cancer agents, including signal transduction inhibitors, have shown great promise in treating hematologic malignancies. However, used as single agents, these drugs may not be curative secondary to innate or acquired cellular resistance. Thus, acute lymphoblastic leukemia (ALL) and other cancer cells may become resistant to rapamycin, an mTOR inhibitor (MTI), following extended exposure to the drug. A strategy to overcome such resistance is to combine targeted agents, and thereby inhibit multiple targets simultaneously. Previously, we have shown activity of MTI in models of both human and murine ALL. In mouse models, treatment of ALL with MTI prolongs survival but may not cure disease. IL-7, a lymphoid growth factor important in the regulation of progenitor B cell development and proliferation, can reverse the inhibitory effects of MTI on human and murine pre-B ALL cells. We wished to further explore the mechanisms by which IL-7-mediated signaling protects ALL cells from the inhibitory effects of MTI, through the investigation of modulators of growth factor signaling in ALL. Thus, we have evaluated the impact of LY294002, an inhibitor of phosphatidyl inositol-3 kinase (PI3K). PI3K is a critical signaling molecule in cell survival and proliferation, with one of its central roles being signal transduction from growth factor receptors to the activation of AKT (an upstream regulator of mTOR). PI3K/AKT pathway over-activation has been implicated in many different cancers. Treatment of ALL cell lines with the PI3K inhibitor LY294002 markedly decreased cell proliferation in a dose-dependent manner. More importantly, the inhibitory effects of LY294002 were additive or synergistic with the inhibitory effects of MTI, and prevented the ability of IL-7 to reverse the inhibitory effects of rapamycin. Treatment of pre-B ALL cell lines with 2.5 μM LY294002 resulted in decreased proliferation to 20–45% of baseline as compared to untreated cells, whereas treatment with a higher dose (5 μM) reduced cell proliferation to 10–20%. Combinations of LY294002 and rapamycin, even at low doses, inhibited cell proliferation to a greater degree than each drug individually. Co-treatment with 2.5 μM LY294002 and low dose rapamycin (1 ng/ml) resulted in profound inhibition of proliferation to <=5%, compared to 20–30% with rapamycin alone. Furthermore, co-treatment with low-dose LY294002 and low-dose rapamycin resulted in greater inhibition than even higher doses of each of these agents individually. While the addition of IL-7 (1 U/ml) to rapamycin-treated cells resulted in the reversal of rapamycin-mediated cell inhibition, the further addition of 2.5 μM LY294002 significantly antagonized this growth factor rescue of MTI-treated ALL cells. The blockade by LY294002 of the IL-7 effect was most apparent in ALL cell lines that were IL-7 dependent, with cell proliferation reduced to <20%. However, the effects were still significant in IL-7 independent cell lines, with proliferation reduced to 20–60%. Similar results were seen using human ALL cell lines. These data suggest that the PI3K signaling pathway serves as a potential rescue pathway from mTOR inhibition, mediating the ability of growth factors to rescue cells from rapamycin;PI3K itself is a therapeutic target for ALL; andcombination therapy with MTI and PI3K inhibitors may be more active than either agent alone.

Overexpression of Ras Homologue Enriched in Brain Like-1 (RhebL1) Leads to Positive Effects on Mouse Hematopoietic Progenitor Cells.

The molecular processes which control self-renewal and differentiation of hematopoietic stem cells (HSCs) are not completely understood. Elucidating these mechanisms and signaling pathways is important in possible ex vivo expansion and treatment of HSCs. It is likely that genes which are highly expressed in HSC populations have important roles in their biology. The stem cell database (http://stemcell.princeton.edu) identified many genes highly expressed in HSC populations using subtractive cDNA technology (Ivanova et al. Science, 2002. 298(5593): p. 601-04). We chose to study the novel gene, Ras homologue enriched in brain like-1 (RhebL1), identified in the stem cell database. Rheb and RhebL1, the two members of the Rheb GTPase family, have positive roles in activating the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway positively affects proliferation, growth and survival of various cell types. Due to its expression pattern and its role in the mTOR pathway, we hypothesized that RhebL1 plays an important role in regulating hematopoietic cell biology. We cloned the full-length RhebL1 cDNA (BC016521) into the bicistronic MIEG3 retroviral vector, which contains the EGFP reporter. We transduced primary mouse bone marrow (mBM) cells with the MIEG3-RhebL1 vector and found positive effects of overexpression of RhebL1 on proliferation and colony-forming ability compared to cells transduced with empty vector MIEG3. Overexpression of RhebL1 increased the proliferation of mBM cells up to 2-fold over control cells in liquid cultures up to one week after transduction. In addition to the positive effect on proliferation, we found that overexpression of RhebL1 in mBM cells significantly increased the absolute number of hematopoietic progenitors, measured by methylcellulose colony assays, in liquid cultures of transduced cells up to one week after transduction. This positive effect on progenitor number was greatest for the CFU-granulocyte-macrophage (GM) progenitors (2.5 fold increase over MIEG3 cells). The number of CFU-GEMM progenitors was also significantly increased by RhebL1 overexpression, while the BFU-Erythroid progenitors were not significantly affected. The transduced cells remained GFP+ during the 6-day methylcellulose colony assay incubation period as determined by confocal microscopy. In addition to the overexpression studies with primary mBM cells, we have expanded our work to involve the BaF3 mouse pro-B cell line. BaF3 cells are dependent on the growth factor IL-3 for survival and proliferation. In addition, the mTOR pathway is involved in IL-3 signaling in these cells (Cruz et al. J. Leuk. Bio., 2005.78(6): p. 1378-85). We found that overexpression of RhebL1 in BaF3 cells increased proliferation after delayed IL-3 addition compared to cells expressing empty vector. Along with an increase in proliferation, BaF3 cells overexpressing RhebL1 had increased mTOR activation as measured by phosphorylated S6 protein levels within the cells during IL-3 starvation and after addition of IL-3. These results indicate that RhebL1 overexpression positively affects the proliferation and colony-formation of primary mouse BM cells. In the BaF3 cell line, RhebL1 overexpression increases mTOR signaling and positively affects the proliferation of the cells after delayed IL-3 addition. Our results suggest a role for RhebL1 in hematopoietic progenitor cell functions.

Mutations of the Catalytic Subunit of Class IA PI3K Are Constitutively Active and Confer Factor-Independent Growth to Early Hematopoietic Cells.

Background: Activating mutations of the catalytic subunit of class IA phosphoinositide 3-kinase alpha (PIK3CA) are clustered in small hot-spot regions of the PIK3CA gene, including exon 9 within the helical domain and exon 11 within the kinase domain. They have been linked to several human neoplasias, including colorectal, breast and hepatocellular cancers. In acute leukemias, PIK3CA mutations have not been investigated in larger chorts and so far only been observed in a few patients. Since the PI3K/Akt/GSK3beta pathway is an important signaling cascade of receptor tyrosine kinases (e.g. Flt3R, Kit) which are frequently activated in acute leukemias we investigated the functional activity of PIK3CA mutants.Materials and methods: We transfected early hematopoietic cells (Ba/F3 cell line) with PI3KCA exon 9 and 11 mutations and investigated the cells in an in vitro factor-independent growth assay and pharmacologic inhibition experiments.Results: We demonstrate that mutations in the helical or kinase domain of PIK3CA lead to the constitutive activation of PI3Kalpha in Ba/F3 cells, inducing factor-independent growth of the IL3-dependent cells. The frequency of IL3-independent Ba/F3 cells after tranfection with exon 9 and 11 PIK3CA mutants was equivalent to the frequency confered by PIK3CA mutants containing the membrane localization signal of either src or ras. Proliferation and survival of the cells were inhibited by the PI3K inhibitors LY294002 and Quercitin or an inhibitor of the PI3K downstream target Akt. Inhibition occurred in a dose- and time-dependent manner and could be reverted by addition of IL-3. One of the major targets of PI3K/Akt signaling is GSK3beta which becomes inactivated after Akt-mediated phosphorylation. By using a GSK3beta-specific inhibitor or LiCl we could show that the inactivation of GSK3beta alone did not result in factor-independent growth of Ba/F3 cells. However, GSK3beta inhibition led to a delay in the induction of cell death after IL3-withdrawal.Conclusion: Activating mutations of PIK3CA, associated with several human neoplasias and acute leukemia are functionally active in hematopoietic cells, confer factor independency and respond to PI3K/Akt inhibition.

Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein.

[Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133.[Methods] Human cord blood CD133-sorted cells were cultured on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, flt-3 ligand, IL-3 and IL-6/soluble IL-6 receptor chimeric protein (FP6) for three weeks, and cultured cells were transplanted into NOD/SCID mice after limiting dilution to calculate the number of SRCs. To confirm whether full multipotency and self-renewal capacity of SRCs were maintained during the culture, cells were transplanted serially into NOD/SCID/γcnull (NOG) mice, and hematopoietic reconstitution was examined. To compare the efficiencies of CD34- and CD133-sorting, we divided each sample into two aliquots and separated CD34+ and CD133+ cells, and calculated the SRC numbers recovered by both separation methods.[Results and discussion] The frequencies of SRCs in the culture-initiating CD133-sorted cells and cultured progeny were calculated as one out of 1,020 and one out of 175 (adjusted to culture-initiating cells), respectively, indicating 6-fold expansion of SRCs that was statistically significant. Delta1 significantly enhanced the expansion rate of SRCs, and addition of IL-3 to this condition further promoted the expansion. In the serial transplantation assays, we found human myeloid and lymphoid reconstitution both in the primary and secondary NOG recipients, verifying the SRC capacity in the cultured cells. Notably, the CD133-sorting was approximately 4.5 times more efficient in collecting SRCs than the CD34-sorting from the same number of mononuclear cells (MNCs) (308 and 254 SRCs by CD133-sorting vs. 67 and 59 SRCs by CD34-sorting from 108 MNCs). Our study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

IL-13 Receptor α2 Promotes Epithelial Cell Regeneration From Radiation–Induced Small Intestinal Injury in Mice

The cytokines interleukin (IL)-4 and IL-13 have pleiotropic effects on a variety of cell types and impact both pathologic changes and tissue remodeling. The aim of this study was to clarify the roles of IL-13 receptor alpha2 (IL-13Ralpha2), which is the high-affinity decoy receptor for IL-13, in gastrointestinal tract epithelial cell turnover and repair. We have compared the regenerative process following mucosal damage induced by whole-body 3-Gy X-ray irradiation of wild-type (WT) and IL-4 receptor alpha gene-deficient (IL-4R(-/-)) mice. Then we treated mice with IL-13Ralpha2 human immunoglobulin (Ig) chimeric protein. Up-regulation of mRNA levels for IL-13 in NK cells in the lamina propria was seen after irradiation of WT mice. Concomitant with vigorous epithelial cell division in the jejunum following irradiation, expression of the IL-13Ralpha2 dramatically increased in myofibroblasts and fibroblasts. In contrast, epithelial cell repair was delayed in IL-4R(-/-) mice, which did not show transient up-regulation of IL-13Ralpha2, although up-regulation of IL-13 was seen. Addition of IL-13 but not IL-4 to primary cultures of small intestine from both WT and IL-4R(-/-) mice induced epithelial cell damage. Treatment of IL-4R(-/-) mice with IL-13Ralpha2-Ig resulted in increased numbers of dividing epithelial cells and improved tissue repair after irradiation. Further, treatment with IL-13Ralpha2-Ig increased numbers of microcolonies of regenerating epithelial cells in the intestine of WT mice after severe damage induced by 12-Gy irradiation. The IL-13Ralpha2 is a major regulatory factor involved in the regeneration of epithelial cells in the gastrointestinal tract.