ALBERT

Description: Abstract 4697 Clinical trials have shown intracoronary or intramuscular injections of hematopoietic stem cells (HSC) ameliorate vascular ischemia. HSCs are isolated from bone marrow (BM), umbilical cord blood (UCB), and adult peripheral blood (AB). Critical limb ischemia patients, injected with autologous HSC into ischemic tissue had improved perfusion and reduced amputations. However, autologous HSC effectiveness declines with age. We hypothesized that UCB CD133+ HSCs’ are more efficacious than autologous HSC at revascularization for an ischemic bed. Immune reactive surface antigens on UCB CD133+ cells were determined using multi-parameter flow cytometry. UCB CD133+ allogeneic immune responses from 72 h co-cultures were analyzed using a mixed lymphocyte culture (MLR) proliferation assays, and induced cytokine production found using TH1(inflammatory)/TH2(anti-inflammatory) cytometric bead assays (CBA) (BD Biosciences (San Jose, CA). In vivo studies were done using NOD/SCID mice treated with low-dose irradiation (200cGy) then femoral ligation. Study mice were divided into 3 treatment groups and underwent intracoronary infusions of; 1×106 UCB CD133+, 0.5×106 UCB mononuclear cells (MNCs), or EGM media alone. Day 3 post-ligation and cell infusion, quadriceps from 3 mice in each group were harvested, total RNA isolated using Trizol™ and RNA for each treatment group pooled. Pooled RNA was converted to cDNA then assayed using mouse ABI Affymetrix™ Gene chip. Computational pathway analyzes were run for significant transcriptome changes. Gene array data was confirmed on select genes using quantitative reverse transcriptase real time PCR (qPCR). Treated mice were followed for ischemic bed reperfusion by Laser Doppler imaging for 28 days post-ligation. UCB CD133+ cells express MHC class I and II, but lack CD80 and CD86. Allogeneic MLR assays using UCB and AB MNCs showed UCB CD133+ cells were stimulatory to MNC proliferation. The MNCs alloresponse to UCB CD133+ cells produced high concentration of interleukin 4 (IL-4) and IL-10 indicative of a TH2 response. These results show allogeneic UCB CD133+ cells induce a TH2 immune response due to an absence of co-stimulation of immune cells. To verify whether the results are recapitulated in vivo, we treated acute limb ischemia in NOD/SCID mice with human UCB CD133+ cells and monitored revascularization by blood flow and Affymetrix mouse gene array. Computational analysis of Affymetrix mouse gene arrays from comparing all 3 treatment protocols indicated that ischemic quadriceps treated with UCB CD133+ cells exhibited a significant enrichment of anti-angiogeneic, chemotaxic, cytokine, and immune response genes. UCB CD133+ treated mice had increase expression of anti-inflammatory, and anti-angiogeneic genes versus controls. Conversely media and UCB MNC treated mice had equal to or higher pro-inflammatory gene expression. The anti-inflammatory gene response was confirmed for 2 of the 3 genes, arginase 1, CXCL7, and tumor growth factor beta binding protein 2 (TGFβBP2) comparing CD133+ treated mice to UCB MNC treated mice. TGFβBP2 was not observed to change in vivo. UCB CD133+ cells treated mice also had improved blood perfusion of the ischemic bed at 28 days post-ligation versus control protocols. The in vivo mouse model supports the in vitro observation, but demonstrates improved reperfusion over controls. Currently there are 2 non-exclusive hypotheses for the function of CD133+ cells in revascularization. First the CD133+ cells integrate and differentiate into new vascular endothelial cells and the second is that the CD133+ cells augment revascularization via a paracrine effect which does not require integration into the host tissue. In vitro studies suggest that UCB CD133+ cells promote an allogeneic TH2 type response because of a lack of co-stimulatory immune surface antigens, resulting in high concentrations of IL-4 and IL-10. This skewed TH2 response inhibits inflammation, the subsequent tissue damage, and may augment revascularization. Although these studies cannot exclude a role for UCB CD133+ cellular integration, results support the paracrine hypothesis as infusion of selected CD133+ cells appears to promote an anti-inflammatory, anti-angiogenic profile at day 3 post-ligation and at 1 month improved reperfusion compared with non-selected MNC. Disclosures: No relevant conflicts of interest to declare.

Description: The reduced expression of nuclear factor of activated T cells-1 (NFAT1) protein in umbilical cord blood (UCB)–derived CD4+ T cells and the corresponding reduction in inflammatory cytokine secretion after stimulation in part underlies their phenotypic differences from adult blood (AB) CD4+ T cells. This muted response may contribute to the lower incidence and severity of high-grade acute graft-versus-host disease (aGVHD) exhibited by UCB grafts. Here we provide evidence that a specific microRNA, miR-184, inhibits NFAT1 protein expression elicited by UCB CD4+ T cells. Endogenous expression of miR-184 in UCB is 58.4-fold higher compared with AB CD4+ T cells, and miR-184 blocks production of NFAT1 protein through its complementary target sequence on the NFATc2 mRNA without transcript degradation. Furthermore, its negative effects on NFAT1 protein and downstream interleukin-2 (IL-2) transcription are reversed through antisense blocking in UCB and can be replicated via exogenous transfection of precursor miR-184 into AB CD4+ T cells. Our findings reveal a previously uncharacterized role for miR-184 in UCB CD4+ T cells and a novel function for microRNA in the early adaptive immune response.

Description: Large granular lymphocyte leukemia (LGL) is chronic clonal lymphoproliferation of CTL. The association of LGL with autoimmune conditions imply that it arises in the context of polyclonal immune response but many features point towards viral etiology. Cytopenias suggest that LGL cells mediate inhibition of hematopoietic progenitors; possible mechanisms include direct cytotoxicity and secretion of inhibitory cytokines. In many aspects, LGL cells resemble normal, terminally differentiated CTL, including lack of CD28 expression, a high content of perforin and granzymes and expression of CD57. Our experiments were designed to gain insight into the gene expression profile of clonal CTL in LGL. First, we analyzed the expression profile of normal mature effector CTL defined by CD57 positivity. Due to low numbers of these cells in the blood of healthy individuals, flow sorted CD57+CTL from 14 controls were pooled. cRNA from patients and pooled controls was hybridized to total human genome U133+ 2.0 microarray covering 47.000 transcripts. For the analysis of LGL gene expression profile we took advantage of VB chain utilization pattern established in previous studies (Wlodarski et al, Blood 2005) and purified malignant LGL based on their VB chain usage and CD57 expression. Each LGL patient harbored an expanded VB clone comprising of 95%, 23% or 83% of the total CTL repertoire (VB1; VB20 and VB13.2, respectively). When expression pattern of LGL cells was compared to that of normal CD8+CD57+ cells, globally, in LGL concordant results were obtained for a total of 761 genes that were differentially expressed ≥2X (259 genes showed an increased expression and 503 were downregulated). Our analysis was in agreement with the previously described features of T-LGL including downregulation of CD28, and overexpression of CD16, FasL, IL8, IL-1Ra, HLA-DR and IL10. Some of the upregulated genes indicate important novel phenotypic differences between LGL and normal CTL cells. For the purpose of this study we focused our analysis on cytokine proteome of T-LGL and found a surprisingly consistent overexpression of transripts encoding cyto-/chemokines (IFN-g, IL-7, IL-8, IL-10, IL-18, CCL2, CCL3, CXCL2, CXCL10, CXCL16). Moreover, LGL clones were characterized by an increased expression of chemokine receptors that are known to facilitate viral infections (CCR1, CCR2). All these changes were described in the context of cellular reaction to intracellular pathogens. Similarly, genes highlighting a nonspecific reaction to stressors (e.g. HSP70) were upregulated in LGL. The elevated expression of IFN-g, Fas-L and IL-18, capable of inhibiting hematopoiesis is consistent with cytopenias seen in the patients studied. Based on the findings of expression arrays, we focussed our analysis on diagnostically useful soluble factors that can be measured in serum and correlated with clinical course. ELISA assays for a selection of cytokines were performed on plasma samples from 24 LGL patients and confirmed their elevated levels in vivo. For the genes for which protein assays were not available (e.g. CCR1, CCR2, CD164, CD302, CD31, CD38, MCL1, TNFRS9) Taqman PCR was performed. Our studies suggest that LGL clones, although phenotypically similar to terminal effector CTL show significantly altered cytokine proteome indicative of an ongoing viral infection. Overexpression of inhibitory cytokines may explain hematopoietic inhibition.

Description: Background: Nuclear factor of activated T cells 1 (NFAT1 or NFATc2) is proposed to plays a role in Graft versus host disease (GVHD) because it regulates the transcription of many cytokines and surface regulatory proteins including: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), IL-13, IFN-γ, CD40L, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α. NFAT1 regulates the expression of these genes through direct interaction with the leucine zipper region of AP-1 (Fos/Jun) and through cooperative binding to a 15-bp DNA sequence that contains both NFAT1 and AP-1 sites. The highly conserved basic regions of AP-1 (Fos/Jun heterodimer) bind to the TGTTTCA consensus sequence in DNA. Human Bach2 is also a basic-leucine zipper (bZip) protein. The amino acid residues in contact with the DNA in the basic regions of c-Jun and c-Fos are also identical in Bach2 (human and mouse). The high conservation of DNA-contacting amino acid residues between Bach2 and AP-1 strongly suggests that Bach2 (homodimers) and AP-1 (Fos/Jun heterodimers) can bind to the same DNA site, which may block the formation of NFAT1/AP-1/DNA complex and the subsequent expression of cytokine genes. Clinical studies have determined that umbilical cord blood (UCB) elicits reduced incidence and severity of GVHD, compared to Bone Marrow (BM), due in part to reduced donor T-cell cytokine production. Methods: We compared the RNA and protein levels of NFAT1, c-Jun, c-Fos, and Bach2 in human UCB CD4+ T-cells with Adult Blood (AB) CD4+ T-cells by Quantitative RT-PCR analyses and Western blots. This comparison was done in both stimulated and non-stimulated conditions. Our ongoing interest is to test whether Bach2 binds the same DNA site as AP-1, and to test the potential of Bach2 and AP-1 competing for the same DNA site using Electrophoretic mobility shift assay (EMSA) with purified Protein of Bach2, c-Jun, and c-Fos from E. coli. Results: Our data showed a high expression of Bach2 RNA in both non-stimulated and stimulated UCB CD4+T-cells. In both the non-stimulated comparison and stimulated comparison samples, we detected no significant changes in RNA levels of NFAT1, c-Jun, or c-Fos. Conclusions: Our data support the hypothesis that Bach2 acts as a transcriptional repressor of cytokine genes due to the fact that highly expressed Bach2 in UCB competes with AP-1 for same DNA binding site.

Description: Clinical benefits of umbilical cord blood (UCB) grafts include lower incidence of acute graft-versus-host disease (aGVHD) following allogeneic transplantation. The dramatically lower expression of the transcription factor Nuclear Factor of Activated T-cells 1 (NFAT1) in UCB-derived CD4+ T-cells compared to adult blood (AB) results in reduced production of inflammatory cytokines following stimulation, in part underlying this advantage. Here we present a novel mechanism by which NFAT1 protein expression is restrained in these cells. Importantly, NFAT1 mRNA (NFATc2) is not significantly reduced in UCB CD4+ T-cells. Gradient separation and cellular fractionation reveal that NFAT1 is indeed translationally repressed in UCB through early stimulation timepoints, which we hypothesized might be due to the activity of microRNA. A search of known microRNA species reveals that miR- 184 is strongly predicted to bind to the NFATc2 3′ untranslated region (UTR). We have confirmed this interaction through luciferase expression and Western Blot analysis. First, luciferase expression vectors containing the short miR-184 binding sequence or the cloned 3′ UTR were constructed and transfected into UCB and AB CD4+ T-cells. In UCB, insertion of the miR-184 binding sequence alone reduced luciferase expression to 38% of control. The cloned 3′ UTR vector exhibited 60% expression compared to control. Both these negative effects were fully reversible by cotransfection with an excess of blocking antisense to miR- 184. Likewise, the miR-184 site did not impact luciferase expression in AB CD4+ T-cells, but expression was reduced by 61% in AB upon cotransfection with miR-184 mimic. miR-184 mimic produced a 23% decrease in luciferase activity from vector containing the NFATc2 3′ UTR. Both these effects were attenuated by cotransfection with an excess of blocking antisense to miR-184. Blocking antisense increased endogenous NFAT1 protein expression in UCB CD4+ T-cells by 86% as measured by quantified Western Blot, without producing a significant change in NFATc2 mRNA quantity. Conversely, miR-184 mimic produced a 31% decrease in NFAT1 protein expression by AB CD4+ T-cells. We confirmed by quantitative RT-PCR (qRT-PCR) that miR-184 exhibited an average of 58.4-times greater expression in UCB than in AB CD4+ T-cells (p=0.005). We have additionally demonstrated via gain-of-function and loss-of-function analyses that the effects on NFAT1 expression by miR-184 are sufficient to affect transcription of the NFAT-dependent pro-inflammatory cytokine IL-2 through 16 hours of in vitro stimulation. This study comprises the first identification of miR-184 activity in primary human lymphocytes and reveals its function in repressing NFAT1 protein expression uniquely in UCB CD4+ T-cells. These findings hold important implications for our further understanding of GVHD, autoimmunity, and neonatal lymphocyte development, and suggest miR-184 as a useful biomarker or target of ex vivo graft analysis.