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Interactions between tumor and immune cells either enhance or inhibit cancer progression. We show here that Stat3 signaling within the tumor microenvironment induces a procarcinogenic cytokine,IL-23,while inhibiting a central anticarcinogenic cytokine,IL-12,thereby shifting the balance of tumor immunity toward carcinogenesis. Stat3 induces expression of IL-23,which is mainly produced by tumor-associated macrophages,via direct transcriptional activation of the IL-23/p19 gene. Furthermore,Stat3 inhibits NF-kappaB/c-Rel-dependent IL-12/p35 gene expression in tumor-associated dendritic cells. Tumor-associated regulatory T cells (Tregs) express IL-23 receptor,which activates Stat3 in this cell type,leading to upregulation of the Treg-specific transcription factor Foxp3 and the immunosuppressive cytokine IL-10. These results demonstrate that Stat3 promotes IL-23-mediated procarcinogenic immune responses while inhibiting IL-12-dependent antitumor immunity. View Publication

Antibody-based therapies,such as rituximab and alemtuzumab,have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas,diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy. View Publication

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac),a part of the core enzyme,has two isoforms,alpha (PP2Ac alpha) and beta (PP2Ac beta),of which PP2Ac alpha is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases,but the mechanisms that control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Ac alpha promoter in human primary T cells. After preparing serially truncated PP2Ac alpha promoter luciferase constructs,we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element motif flanked by three GC boxes. Shift assays revealed that CREB/phosphorylated CREB and stable protein 1 could bind to the region. Furthermore,we demonstrated that methylation of deoxycytosine in the CpG islands limited binding of phosphorylated CREB and the activity of the PP2Ac alpha promoter. In contrast,the binding of stable protein 1 to a GC box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine,a DNA methyltransferase inhibitor,showed increased expression of PP2Ac alpha mRNA. We propose that conditions associated with hypomethylation of CpG islands,such as drug-induced lupus,permit increased PP2Ac expression. View Publication

BACKGROUND: Infection with HIV-1 has been shown to alter expression of a large array of host cell genes. However,previous studies aimed at investigating the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines. To better approximate natural conditions,we monitored gene expression changes in a cell population highly enriched in human primary CD4+ T lymphocytes exposed to HIV-1 using commercial oligonucleotide microarrays from Affymetrix. RESULTS: We report here that HIV-1 influences expression of genes related to many important biological processes such as DNA repair,cellular cycle,RNA metabolism and apoptosis. Notably,expression of the p53 tumor suppressor and genes involved in p53 homeostasis such as GADD34 were up-regulated by HIV-1 at the mRNA level. This observation is distinct from the previously reported p53 phosphorylation and stabilization at the protein level,which precedes HIV-1-induced apoptosis. We present evidence that the HIV-1-mediated increase in p53 gene expression is associated with virus-mediated induction of type-I interferon (i.e. IFN-alpha and IFN-beta). CONCLUSION: These observations have important implications for our understanding of HIV-1 pathogenesis,particularly in respect to the virus-induced depletion of CD4+ T cells. View Publication

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis,type 1 diabetes mellitus,and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5),containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin,is a potent and selective blocker of these channels. However,a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability,ShK-192,contains a nonhydrolyzable phosphotyrosine surrogate,a methionine isostere,and a C-terminal amide. ShK-192 shows the same overall fold as ShK,and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg,approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24,48,and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells. View Publication

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally,copy number losses are identified using metaphase cytogenetics,whereas detection of UPD is accomplished by microsatellite and copy number analysis and as such,is not often used clinically. Recently,introduction of single nucleotide polymorphism (SNP) microarrays has allowed for the systematic and sensitive detection of UPD in hematologic malignancies and other cancers. In this study,we have applied 250K SNP array technology to detect previously cryptic chromosomal changes,particularly UPD,in a cohort of 301 patients with myelodysplastic syndromes (MDS),overlap MDS/myeloproliferative disorders (MPD),MPD,and acute myeloid leukemia. We show that UPD is a common chromosomal defect in myeloid malignancies,particularly in chronic myelomonocytic leukemia (CMML; 48%) and MDS/MPD-unclassifiable (38%). Furthermore,we show that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations,including newly identified missense mutations in the proto-oncogene c-Cbl in 7 of 12 patients with UPD11q. Acquired mutations of c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process in a subset of MDS/MPD,including CMML. View Publication

The CD31(+) subset of human naive CD4(+) T cells is thought to contain the population of cells that have recently emigrated from the thymus,while their CD31(-) counterparts have been proposed to originate from CD31(+) cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4(+) T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31(+) naive CD4(+) T cells from adult peripheral blood compared with the CD31(-) subset. IL-7-driven proliferation did not result in loss of CD31 expression,suggesting that CD31(+) naive CD4(+) T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore,IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together,our data suggest that during adulthood CD31(+) naive CD4(+) T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. View Publication

Autoimmune pathologies are caused by a breakdown in self-tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self-tolerance in an antigen-specific manner. Studies about tolDC have focused largely on generating stable maturation-resistant DC,but few have fully addressed questions about the antigen-presenting and migratory capacities of these cells,prerequisites for successful immunotherapy. Here,we investigated whether human tolDC,generated with dexamethasone and the active form of vitamin D3,maintained their tolerogenic function upon activation with LPS (LPS-tolDC),while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS-tolDC,but not tolDC,expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore,LPS-tolDC were superior to tolDC in their ability to present type II collagen,a candidate autoantigen in rheumatoid arthritis. tolDC and LPS-tolDC had low stimulatory capacity for allogeneic,naïve T cells and skewed T cell polarization toward an anti-inflammatory phenotype,although LPS-tolDC induced significantly higher levels of IL-10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen-presenting activity in tolDC is important for optimizing their therapeutic potential. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that,in most transplantation patients,variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype,as well as through phenotypic and functional analyses of NK cells,both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells,including patient leukemia blasts. Differently,KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly,this was due to recognition of C2 by KIR2DL2/3,as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably,however,C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether,these results may have important clinical implications for the selection of optimal donors for haplo-HSCT. View Publication

Interleukin-15 (IL-15) is essential for natural killer (NK) cell differentiation. In this study,we assessed whether the receptor tyrosine kinase Axl and its ligand,Gas6,are involved in IL-15-mediated human NK differentiation from CD34(+) hematopoietic progenitor cells (HPCs). Blocking the Axl-Gas6 interaction with a soluble Axl fusion protein (Axl-Fc) or the vitamin K inhibitor warfarin significantly diminished the absolute number and percentage of CD3(-)CD56(+) NK cells derived from human CD34(+) HPCs cultured in the presence of IL-15,probably resulting in part from reduced phosphorylation of STAT5. In addition,CD3(-)CD56(+) NK cells derived from culture of CD34(+) HPCs with IL-15 and Axl-Fc had a significantly diminished capacity to express interferon-gamma or its master regulator,T-BET. Culture of CD34(+) HPCs in the presence of c-Kit ligand and Axl-Fc resulted in a significant decrease in the frequency of NK precursor cells responding to IL-15,probably the result of reduced c-Kit phosphorylation. Collectively,our data suggest that the Axl/Gas6 pathway contributes to normal human NK-cell development,at least in part via its regulatory effects on both the IL-15 and c-Kit signaling pathways in CD34(+) HPCs,and to functional NK-cell maturation via an effect on the master regulatory transcription factor T-BET. View Publication

Many cellular responses,such as autoimmunity and cytotoxicity,are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here,we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk,concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore,Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity,providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex,observed here with two types of inhibitory receptors,expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism. View Publication