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The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, we have generated a series of deletion mutants and have investigated their trans-activation potential as well as their DNA-binding activity. Our results have allowed us to delineate the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218–319) are centred on a sequence rich in charged amino acids (residues 259–281). A region (residues 320–482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues markedly inhibits specific DNA binding. Finally the last 110 residues of A-Myb (residues 643–752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. We have also investigated the functional interaction between A-Myb and the nuclear adaptor molecule CBP [cAMP response element-binding protein (CREB)-binding protein]. We demonstrate that CBP synergizes with A-Myb in a dose-dependent fashion, and that this co-operative effect can be inhibited by E1A and can also be observed with the CBP homologue p300. We show that this functional synergism requires the presence of the A-Myb charged sequence and that it involves physical interaction between A-Myb and the CREB-binding domain of CBP.

PTX3 is a prototypic long pentraxin composed of a C-terminal domain similar to those of classical pentraxins (e.g., C reactive protein) and an unrelated N-terminal portion. PTX3 is expressed in a variety of cell types, notably mononuclear phagocytes and endothelial cells, after exposure to the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The present study was designed to assess whether mycobacterial components were able to induce expression and production of PTX3. Mycobacterial lipoarabinomannan (LAM) induced expression of PTX3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside. Among mononuclear cells, monocytes were responsible for LAM-induced PTX3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced PTX3 expression. Pretreatment with actinomycin D abolished LAM-induced PTX3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced PTX3 expression was associated with the production of immunoreactive PTX3. IL-10 and IL-13 did not inhibit the induction of PTX3 by LAM. Under the same conditions, these anti-inflammatory cytokines inhibited MCP-1 expression. In contrast, gamma interferon inhibited LAM-induced PTX3 expression. Thus, in addition to IL-1, TNF, and lipopolysaccharide, mycobacterial cell wall components also induce expression and production of the long pentraxin PTX3. The significance of PTX3 in the immunobiology of mycobacterial infection and its relevance in relation to clinical involvement remain to be determined.

Abstract With limitation to the “proinflammatory program” induced in endothelial cells by exposure to interleukin-1, tumor necrosis factor, and interleukin-6, we review the available data on the signaling for these three cytokines, from receptor engagement to induction of gene transcription. Only a few molecular pathways have been characterized so far, and key issues in endothelial biology, such as endothelial specificity of gene expression and heterogeneity of different endothelial populations, remain largely unexplored.

Monocyte chemotactic protein-1 (MCP-1) interacts with the chemokine receptor CCR2. Two CCR2 cDNAs have been described. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated natural killer (NK) cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. We found that bacterial products and cytokines affect CCR2 expression. Interleukin-2 (IL-2) augmented CCR2 mRNA in monocytes and NK cells. The augmented migratory capacity of Reactivated versus resting NK cells was associated with increased CCR2 transcript levels. Lipopolysaccharide (LPS) and other microbial agents caused a rapid and drastic reduction of CCR2 mRNA levels. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced. These results suggest that regulation of receptor expression, in addition to agonist production, is probably a crucial point in the regulation of the chemokine system. Down-regulation of chemokine receptor expression may play a role in the modulation of HIV infection in macrophages by LPS. Levels of MCP-1 were markedly elevated in the cerebrospinal fluid (CSF) but not in blood of HIV-infected patients with cytomegalovirus (CMV) encephalitis. The CSF levels of MCP-1 in CMV encephalitis were markedly higher than those found in the CSF of HIV-infected patients with or without unrelated neurological diseases. IL-8, the prototype of C-X-C chemokines and RANTES and macrophage inflammatory protein-1α (C-C chemokines) were not substantially increased in the liquor of CMV encephalitis patients. High levels of MCP-1 may underlie monocyte recruitment and tissue damage in CMV encephalitis and may represent a rapid and useful tool in the diagnostic armamentarium for neurological disorders associated with HIV.

Background Adult stem cells, expecially those of mesodermal origin (MSCs), have received attention as an ideal source of regenerative capable cells because of their multipotentially and ability to replicate. Regenerative cells from the bone marrow, peripheral blood, and umbilical cord blood have been used to treat a variety of diseases. Adipose tissue is an attractive source of adult stem cells due to its abundance and surgical accessibility. Recent studies have shown the efficacy of the autologous fat grafting in the treatment of different skin diseases including localized scleroderma and ischemic diseases. Objectives We characterized phenotypically and functionally adipose tissue-derived mesenchymal stem cells (ADMSCs) from patients with Systemic Sclerosis (SSc). Methods Human adipose tissue samples were obtained from 10 patients with the diffuse form of SSc (dcSSc) undergoing autologous lipostructure for the treatment of fibrotic perioral changes. As controls, adipose tissue samples were obtained from 10 healthy donors (HD) undergoing aesthetic surgery. Lipoaspirate tissue was collected from elective liposuction procedures and processed by both explant and enzymatic methods. Key parameters of ADMSCs function and phenotype were assessed including the ability to: express cell surface antigens defining the MSC population (CD105, CD73, CD29, CD90, CD44 marker profile was examined by FACS analysis), proliferate (growth kinetics assay), differentiate along the adipogenic and osteogenic lineages, suppress in vitro lymphocyte proliferation induced by the mixed lymphocyte reaction. Results Cultured ADMSCs obtained from the processing of adipose tissue showed no significant differences in morphology and in proliferation rate in the culture conditions used. Phenotypically, SSc ADMSCs highly expressed CD105, CD73, CD90, HLA-ABC and were mostly negative for HLA-DR expression. The absence of contaminating hematopoietic stem cells was confirmed by the negative expression of CD34, CD14 and CD45 antigens in all studied cases. No phenotypic differences were observed between ADMSCs from patients with SSc and controls. When cultured in standard induction medium, all SSc and HD ADMSCs differentiated toward the osteogenic and adipogenic lineages. In MLR assays, no significant differences in ADMSCs mediated inhibition of proliferation were observed between SSc patients and controls. Conclusions Autologous adipose graft could be a new therapeutic option for the treatment of skin diseases such as SSc. The mechanisms through which adipose tissue exerts its therapeutic potential in tissue repair is not yet fully defined, but it might be related to the well known ability of MSCs to modulate inflammatory and autoimmune process, secrete soluble factors capable to stimulate functional recovery of injured cells and differentiate into other cell types. The results of our study show that ADMSCs from patients with SSc exhibit the same phenotypic, proliferative, differentiation potential and immunosoppressive properties as HD. Further studies are need to clarify the ADMSCs specific contribution to the clinically relevant benefits in SSc soft-tissue reconstruction. Disclosure of Interest None Declared

We investigated whether clinically achievable concentrations of the histone deacetylase (HDAC) inhibitors givinostat and hydroxyurea induce synergistic cytotoxicity in Jak2(V617F) cells in vitro and through which possible mechanism. Givinostat and hydroxyurea at low doses potentiated the pro-apoptotic effects of each other in the Jak2(V617F) HEL and UKE1 cell lines. Givinostat induced 6.8%-20.8% and hydroxyurea (HU) 20.4%-42.4% cell death alone and 35.8%-75.3% in combination. The effect was statistically significant using the median effect Chou-Talalay method, resulting in a combination index less than 1, indicating synergy. Givinostat alone induced cell cycle arrest of the cell lines in G0/G1 and hydroxyurea in S phase, whereas both drugs together led to a G1 block. At the molecular level, hydroxyurea counteracted the induction of p21CDKN1A by Givinostat and potentiated caspase 3 activation, explaining at least in part the increased apoptosis observed in presence of both compounds. We also verified the effect of the same drugs in colony assays of freshly isolated Jak2(V617F) polycythemia vera cells. In this case, low doses of the compounds were additive to each other. These results suggest that combined treatment with givinostat and hydroxyurea is a potential strategy for the management of Jak2(V617F) myeloproliferative neoplasms.

Free radicals play an important role in inflammation. We found that reactive oxygen intermediates (ROI) inhibit interleukin-1beta (IL- 1beta) binding on human myelomonocytes. Production of superoxide anion (O2-) by Xanthine (X) and Xanthine-Oxidase (XO) or NADPH caused a reduction (48% +/- 15% in 25 experiments) in the IL-1beta binding of polymorphonuclear cells (PMN) and monocytes that was inhibited by superoxide dismutase (SOD). Hydrogen peroxide (H2O2) was only active on monocytes and this effect was prevented by catalase. O2(-)-induced loss of IL-1beta binding on PMN reached half maximum at 5 minutes and peaked after 30 minutes. The reduction of IL-1beta binding was due to reduction of IL-1beta receptors (R) on PMN surface without any change in affinity. ROI-induced reduction of surface IL-1R was not caused by receptor internalization, but rather by the release of a soluble form (45 kD) of the type II decoy R. The action of ROI on IL-1 binding was selective because major histocompatibility complex class I, CD18 and CD16 were unaffected. The O2(-)-induced release of IL-1 decoy R was not affected by protein synthesis inhibitors, but was partially blocked by protease inhibitors. Release of the IL-1 type II decoy R might represent one mechanism by which ROI antagonize and limit the proinflammatory effects of IL-1.

The myb family of genes include the virally encoded v-myb oncogene, its normal cellular equivalent c-myb and two related members called A-myb and B-myb. They are all transcription factors that recognize the same DNA sequence (PyAACG/TG) and are all involved in the regulation of proliferation and differentiation in different cell types, including hematopoietic cells. C-myb is most highly expressed in hematopoietic cells and its oncogenic activation leads to transformation of these cells. Several lines of evidence have demonstrated that c-myb regulates both the proliferation and differentiation of hematopoietic cells of different lineages. The mechanisms of action of c-myb and v-myb are becoming clearer, mostly through the study of the different genes that are regulated by these transcription factors and the cofactors with which c-myb and v-myb co-operate. More recently the biological and biochemical functions of the B-myb and A-myb gene products have been investigated. Evidence for the function of the different members of the myb family in relation to hematopoietic proliferation and differentiation is presented, and the different roles of the myb genes are discussed.

The IL-1 system includes 2 agonists, alpha and beta, processing and transport molecules, receptor antagonists, signalling receptor, a decoy receptor and an accessory molecule. Negative pathways of regulation include the antagonists, of which 3 isoforms have been cloned and the type II "decoy" receptor. Molecules that regulate inflammation and immunity coordinatively affect different components of the system. The complexity of the system and the existence of unique pathways of negative regulation, the antagonists and the decoy receptor, emphasize the need for a tight control of the production and action of IL-1.

Endothelial cells play an active role in several different inflammatory and immunological reactions which take place in the tissues.

Abstract 743 Background: Acute Graft versus host disease (aGvHD) is a severe complication of allogeneic hematopoietic stem cell transplantation (HSCT). Conventional treatment with high dose steroids fails to achieve a complete and sustained response in more than 50% of patients. Several second line treatments have been described but none of these can be considered superior or a standard of care (Paul J. Martin et al, BBMT 2012). Among these treatments, the use of third party mesenchymal stromal cells (MSC) has been proposed (LeBlanc et al, Lancet 2008). In this study, we assessed the safety and efficacy of third party human MSC, in a prospective, multicenter, phase I study (EudraCT 2008–007869-23). Methods: Forty-seven patients with steroid-resistant, acute or chronic grade II-IV GvHD were enrolled into this study. Human MSC were obtained from bone marrow harvests of healthy donors and expanded in vitro using serum free medium supplemented with human platelet lysate (Capelli C et al, BMT, 2007; Capelli C. et al, Cytotherapy 2009). In vitro expanded MSC were produced in two officially authorized Cell Factories and tested in four Italian Hematology Units. The primary endpoint of this study was the safety. Secondary endpoints were the response of GvHD (evaluated 28 days after the last MSC infusion), as well as the overall survival and transplant-related deaths. Blood samples were periodically collected before and after MSC infusion to measure plasma levels of IL2Ralpha by ELISA, as previously described by our group (Dander E et al, Leukemia 2012). Results: Between August 2009, and June 2012, 47 patients (16 children, 31 adults, median age 25.5 years, range 1 to 67) were treated. The median dose of infused MSC was 1.5×106 cells per kg bodyweight. Enrolled patients presented with aGvHD in 37 cases, chronic overlap syndrome in 7 cases, and chronic classic GvHD in 3 cases. Fifteen pts had grade II GvHD, 23 grade III and 9 grade IV, according to NIH criteria. In 17 cases GvHD involved a single organ, in 24 cases 2, and in 6 cases 3 organs. Prior to MSC infusion 22 patients had received only high dose steroids, 12 patients received one cycle of pentostatin (1 mg/kg bodyweight for 3 days, Schmitt T. et al BMT, 2011: 46 580–585), while 13 received other conventional immunosuppressants. Patients received a median of 3 MSC infusions (range 1 to 8). No side effects were registered immediately after MSC infusion and no complications were lately referred as MSC-related. Overall, in 30 patients (63.8%) a clinical response of GvHD was registered. Thirteen of these patients (27.6%) had a complete response and 17 (36.1%) a partial response to treatment. Twenty-two of the 30 responding patients did not require further lines of immunosuppression after MSC infusion. Response was significantly more likely in patients exhibiting grade II GvHD versus those exhibiting more severe gradings (87.5% vs. 51.6%, p = 0.02) and in patients receiving MSC in a time interval of 30 days from the onset of GvHD (75.9% vs. 43.7%, p= 0.05). Current median follow up for this cohort is 200 days (range 30–1066). Responders show a significant lower transplant-related mortality (10.0% vs. 88.2%, p Measurements of plasmatic levels of IL2Ralpha, when comparing responders vs non-responders patients, showed a statistically significant difference in terms of fold decrease of the marker (p=0.027), corroborating clinical results. Similarly, a significant trend of fold decrease change (p=0.058) was observed when comparing responding patients receiving MSC within or after 30 days from the onset of the disease, in line with clinical results. Conclusions: This study confirms that human MSC prepared in academic cell therapy facilities may represent a safe and effective treatment of patients with steroid-refractory GvHD. Plasmatic inflammatory markers may help in evaluating and monitoring of clinical response. The sequential or combined administration of MSC and other immunosuppressants, such as pentostatin, is equally safe and feasible and deserves further investigation. We suggest to consider the use of MSC promptly, as early as possible, after steroid failure. Disclosures: No relevant conflicts of interest to declare.

By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.

Abstract 657 Background: Cytokine Induced Killer (CIK) cells have been previously shown by us and others to possess non restricted, NK-like anti-tumoral cytotoxicity in vitro and in vivo, with little graft-versus host disease (GVHD), in vitro and in several animal models. We have previously performed a phase I study with donor derived CIK cells in 11 adult hematologic patients and shown that the infusions of CIK cells (median 12.4 × 106/kg, range 7.2 to 87.4) were well tolerated and no acute or late infusion related reactions were recorded. Acute GVHD (grade I and II) was observed in 4 patients and progressed into extensive chronic GVHD in two cases (Introna et al, Haematologica 92:952, 2007). Methods: This study is an open-labeled, multicenter, exploratory phase IIA study to evaluate the safety (dose-finding) and efficacy of a sequential administration of donor derived unmanipulated lymphocytes (DLI) plus in vitro expanded CIK cells to patients with hematologic malignancies relapsing after related or unrelated allogeneic hematopoietic stem cells transplantation. The protocol was formally approved by the italian competent national authority (Agenzia Italiana del Farmaco, AIFA) on 14/04/2009. Two infusions of unmanipulated DLI (1×106/kg each) were given with a minimum interval of 3 weeks. Three infusions of donor CIK cells were then administered according to a dose escalating program, starting 3 weeks after the second DLI. CIK administrations were separated by 3 weeks intervals. Up to 4 combinations of dose escalating levels were provided in sequential order until the maximal tolerated dose (MTD) was reached. Indeed the first triplet of patients was supposed to receive CIK cells at the doses of 1×106/kg, 1×106/kg and 5×106/kg, the second 1×106/kg, 5×106/kg, 5×106/kg, the third 1×106/kg, 5×106/kg,10×106/kg, the last triplet 5×106/kg, 5×106/kg, 10×106/kg. In case of grade II or more severe acute GVHD, the next scheduled infusion was planned to be suspended. Only grade IV acute GVHD was considered the dose limiting toxicity (DLT). Once identified the MTD, this same dose will be administered up to 24 patients in a two-stage Simon's design. Results: So far 16 patients have been enrolled and 12 are evaluable. No DLT (grade IV acute GVHD) was observed at any dose. One patient suffered from grade III skin and gut acute GVHD in the group which received the highest dose and the therapy was stopped after the first administration of CIK cells (5×106/kg), while another patient showed grade I skin acute GVHD in this same group, but completed the 3 administrations program. So far, 7 patients have received the highest dose of CIK cells and they all completed the schedule without toxicity except one case. As per protocol, the best clinical response was evaluated 100 days after the end of the last CIK administration. We observed 3 CR (3 AML), 5 PR (3 AML, 1 MM and 1 HD) and 4 NR (2 AML, 1 NHL and 1 MM) in the 12 evaluable patients. Of the 12 evaluable patients, 5 required additional therapy at the end of the cell therapy program because of NR (3 patients) or to improve the PR (2 patients). Four patients died after a mean 63 days (3 due to relapse and 1 for infection), 6 patients are in PR and 2 patients remain in CR. Conclusions: These preliminary observations suggest that the sequential infusion of DLI and CIK cells is feasible with relatively minor toxicity, the MTD has not been achieved and the triplet of three CIK administrations of 5×106/kg, 5×106/kg and 10×106/kg will be further evaluated in 17 additional patients. This cell therapy program confirms the antineoplastic activity of this cell therapy schedule in some patients relapsing after allogeneic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

The ontogeny and function of white blood cells require an intimate relationship with vascular endothelium. In the bone marrow, endothelial cells (EC), important producers of molecules with colony-stimulating (CSF) activity, are one crucial constituent of the hematopoietic milieu. In tissues, inflammatory and immunological reactions involve the active participation of EC, which interact closely with leukocytes.

Abstract 797 We have previously shown that the pan-HDAC inhibitor ITF2357 has strong cytotoxic activity against cells from patients with myeloproliferative neoplasms (MPN) bearing JAK2 mutation at position 617. Indeed ITF2357 inhibited colony growth of JAK2V617F positive cells at doses 5–10 fold lower than those required to block JAK2 wild type cells. We have therefore investigated here the molecular mechanism of this effect. Three cell lines homozygotes (HEL, UKE1) or heterozygotes (SET2) for the JAK2V617F mutation were used along with cell lines bearing JAK2 wild type (K562 and KG1). We confirmed the higher sensitivity of mutated with respect to unmutated cell lines in colony formation assay (mean IC50 42 nM versus 179 nM) and alamar blue assay (mean IC50 84 nM vs 325 nM, respectively). In proliferation assays measuring number of live and dead cells at different time points, we observed that 100 nM ITF2357 blocked the proliferation of both JAK2 mutated and unmutated cell lines to a similar extent, with mean inhibition of 31–69% at 72 hours, but induced apoptosis more efficiently in JAK2 mutated (mean 34%) versus unmutated cells (mean 2%). By cell cycle analysis we could show a block in G1 phase of cell cycle in JAK2V617F cells treated with 100 nM drug. In order to unravel the mechanism of specific inhibition of JAK2 mutated cells by ITF2357, we first investigated expression of HDAC isoforms in the different cell lines. We could detect HDAC1, HDAC2 and HDAC3 proteins in Western blots but these were not differentially expressed in a panel of 3 JAK2 mutated and 3 wild type cell lines. We then set out to analyse the molecular mechanism of action of ITF2357 by global gene expression analysis. Using the Rank Product method with a false positive prediction (pfp) of 0.05 and a 2 fold change cut off parameters, we observed 716 and 863 genes modulated at 6 hours by 250 nM ITF2357 in HEL and UKE-1 cell lines, respectively; 293 of these, (179 up- and 114 down-regulated), were common between both cell lines and 10 were subsequently validated by Q-RT-PCR. Among differentially expressed genes, a number are known to play an important role in the control of proliferation and /or apoptosis, most notably APAF1, BCL2L11, CCNG2, NFKB2, MXD1 and TP53INP1, while additional 6 genes (C-MYB, A-MYB, TAL1, NFE2, MLF1, NOTCH2) are involved in the control of hematopoietic differentiation. Of particular interest is NFE2, which was down modulated 2.7 fold by ITF2357 at 6 hours at the RNA level and by about 2 fold at 24 hours at the protein level. NFE2 has been reported to be hyperexpressed in JAK2V617 MPN patients. We also showed that ITF2357 downmodulated NFE2 expression 2 fold also in CD34+ cells purified from these patients. Given the accepted role of NFE2 in the control of erythroid progenitor cell proliferation and differentiation, and its enhanced expression in MPN patients, our data suggest that NFE2 down-regulation by ITF2357 may at least partially explain the drug effect on growth of MPN progenitor cells. The regulation of NFE2 expression and that of other hematopoietic transcription factors and regulatory proteins in response to ITF2357 is under investigation in our laboratory and data will be presented. Disclosures: Fossati: Italfarmaco SpA: Employment. Rambaldi:Italfarmaco SpA: Research Funding. Golay:Italfarmaco SpA: Research Funding.

Abstract 2306 In the last few years the usage of third party mesenchymal stem cells (MSC) as therapy for steroid-refractory Graft versus Host Disease (GvHD) is constantly increasing and holds big promises. Nevertheless, at our knowledge, studies on MSC efficacy have been scarcely corroborated by biological analysis of patient response to cell infusion. Here, we report the immunological monitoring of 8 patients (7 male, 1 female; aged 4 to 33 years), with steroid-refractory GvHD (grade II to III), who received MSCs, between August 2009 and June 2010. GvHD presented as acute in 6 cases and chronic in 2 cases. In 5 cases GvHD occurred as a single organ pathology (2 skin, 2 gut, 1 liver), while in 3 cases GvHD had multi-organ involvement (1 liver and oral mucosa, 1 skin and oral/ocular mucosa, 1 skin, gut and liver). All patients received 2 to 3 MSC infusions from third party donors aiming at 1 × 106/kg recipient body weight MSCs for each infusion. After MSC therapy, 2 patients showed complete response, 3 patients showed partial response, whereas 3 patients did not respond to MSC infusion. To better comprehend the immunomodulatory effects of MSC infusions, we studied GvHD plasmatic markers, inflammatory cytokines and CD4+ T-cell subsets circulating in the peripheral blood (PB) of enrolled patients before MSC infusion and at day 7, 14 and 28 after cell therapy. In accordance with clinical observations, in patients responding to MSC infusions, we observed a dramatic decrease of three validated GvHD plasmatic markers TNFRI, IL2Rα and elafin (Paczesny S et al. Blood 2009) to the mean levels of Healthy Donors (HD). In particular, at day 28 after therapy, TNFRI and IL2Rα levels decreased of 2 times (range=1.9-2.4 and range=1.4-2.8, respectively) and elafin levels decreased of 2.5 times (range=1.7-3.6). Partially responding patients showed a transient decrease of TNFRI, IL2Rα and elafin levels, while non responding patients showed stable or even increasing levels of all analysed markers. Moreover, we investigated the effect of MSC infusion on lymphocyte counts. We demonstrated that patients responding to MSC infusion, oppositely to non responders, strongly decreased total and CD4+ lymphocyte counts in the PB (mean total T-cell Fold Decrease (FD)=11.85, range=1.3-116; mean CD4+ T-cell FD=12, range=1.5-116). Interestingly, after MSC infusion, CD4+ T-cell subsets changed significantly: Tregs increased and Th1 and Th17 populations decreased, and a new CD4+ cell subset balance was observed starting from day 7 after therapy. In particular, the mean FD of Th1/Treg ratio was 4.1 (range=4-4.2) and the mean FD of Th17/Treg ratio was 4.7 (range=3.3-6). Correspondingly, patient symptoms also gradually improved, suggesting an association between GvHD clinical course and CD4+ T-cell imbalance, reverted by MSCs in responding patients. In partially responding patients Th1/Treg and Th17/Treg showed a transient decreased and even slightly increased in the case of non responding patients. In accordance with the decrease of Th1 CD4+ T cells in the PB of patients responding to MSC infusion, we observed a valuable decrease of IFNγ plasma concentrations (mean FD=48, range=30-65 in complete responders), which reached the levels typical of HD. In summary, despite its limited size, the present study suggests that MSCs, upon infusion, are able to convert an inflammatory environment to a more physiological one, both at a cellular level, promoting the expansion of circulating Tregs, and at a molecular level, diminishing inflammatory cytokines. Further studies on a larger group of patients, clarifying the mechanisms of action used in vivo by MSC to tune ongoing allo-reactions, will be fundamental to provide the rationale for improving current clinical trials. Disclosures: No relevant conflicts of interest to declare.

Abstract 4285 Cytokine induced killer (CIK) cells are immune-effector cells that can be expanded in vitro in presence of rhIL-2, starting from peripheral blood mononuclear cells stimulated by interferon-γ and anti-CD3 antibody. CIK cultures at the end of in vitro expansion contain a mean of 40–75% CD3+CD56+ CIK cells, 20–60% CD3+CD56- T cells and 1–10% CD3-CD56+ NK cells. They show MHC-unrestricted cytotoxicity towards neoplastic but not normal targets. Their ease of production in vitro and anti-tumor potential have made them suitable candidates for cell therapy programs in solid and hematopoietic tumour treatment. CIK cells have shown cytotoxic activity in vitro against hematopoietic neoplastic cells, including B Non-Hodgkin's lymphoma (B-NHL). Other biological treatments available for B-NHL are the anti-CD20 antibodies such as type I Rituximab and a new generation glycoengineered type II GA101 antibody. These antibodies are thought to act mostly through immune mediated mechanisms including phagocytosis, complement mediated cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). GA101 has reduced CDC activity compared to type I anti-CD20 antibodies such as Rituximab. In addition, GA101 was defucosylated in its Fc portion to mediate increased ADCC. We have investigated the possibility of combining adoptive immunotherapy by Cytokine Induced Killer (CIK) cells with anti-CD20 type I Rituximab and type II GA101mAb, to optimize B-NHL therapy. CIK cultures alone demonstrated significant cytotoxic activity against a panel of B-NHL cell lines or freshly isolated samples, in either an autologous or allogeneic combination (26-27% killing at 30:1 ET ratio). This natural cytotoxic activity was mainly due to the predominating CD3+CD56+ CIK population (40-75%) present in the cultures. The addition of anti-CD20 mAbs increased CIK mediated cytotoxicity versus B lymphoma target cells and major enhancement was observed with GA101 compared to Rituximab (respectively 34% versus 16% increased lysis at 10:1 E:T ratio). This enhancement was mainly due to ADCC mediated by the small NK cell fraction (1-10%) present in CIK cultures because NK depletion by CD5 immunoselection at the end of expansion did not abolish the basal natural cytotoxicity of CIK cultures but abolished the enhancement observed in presence of anti-CD20 antibodies. The activation of NK cells in CIK cultures, evaluated by CD107a mobilization, was much more effective using GA101 rather than Rituximab (respectively 28% versus 19% CD107a+, p Disclosures: Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Rambaldi:Roche: Honoraria. Golay:Roche: Honoraria. Introna:Roche: Honoraria.

The levels of expression of the murine c-myb gene, like those of several other proto-oncogenes, can be controlled by a block of transcriptional elongation within the first intron of the gene. We have performed run-off experiments with double- and single-stranded probes on the myelomonocytic cell line U937, and show that this mechanism of transcriptional arrest is true also for the human c-myb gene and takes place within the first intron. Furthermore, we have sequenced the entire first intron of the human c-myb gene, and discuss the sequence structure in relation to its putative ability to arrest RNA polymerase II and its high degree of homology with the equivalent murine intron.

Abstract 3024 Poster Board II-1000 Cytokine induced killer (CIK) cells cultures can easily be obtained by stimulating PBMCs with monoclonal antibody anti-CD3 OKT3, IFNgamma and IL2. After 3-4 weeks at least 3 separate populations are present in the culture: CD3+/CD56-/CD8+ precursors (40.5 ± 19.9%), CD3-CD56+ NK cells (2.5 ± 1.5%) and CD3+/CD56+/CD8+ CIK cells (56.9 ± 21%) which show a T EMRA phenotype (Franceschetti et al., Exp Hematol. 37, 616-628, 2009). CIK cultures are currently used in allogeneic or autologous settings as potential anti-neoplastic effectors for adoptive transfer clinical approaches. We have further characterised the mechanism of target cell recognition and role of activating receptors and of lytic mediators in the cytotoxicity of purified CD3+/CD56+/CD8+ CIK cells. We have observed that CIK cells can kill targets through at least two distinct mechanisms: the first TCR-dependent and antigen-specific and the second non TCR-dependent. Indeed, upon TCR/CD3 crosslinking in CIK cells we observe ERK-1/2 phosphorylation, IFNgamma production (mean 32.6% of positive cells by intracellular staining) and TNFalpha production (mean 19.6%). CD3 ligation by OKT3 results in a significant increase over time in the percentage of CIK cells undergoing degranulation evaluated as CD107a positive cells (respectively 15.5 ± 2.2% at 60', 24.4 ± 1% at 120', 32,9 ± 8.7% at 180' and 34.2 ± 11.1% at 240'). CD3 ligation on CIK cells can induce cytotoxicity in a reverse Ab-dependent killing assay. Addition of OKT3 enhances also the cytotoxicity of CIK cells against K562 leukaemic target (from 16 ± 5% to 50 ± 4 %, E:T 10:1; p Disclosures No relevant conflicts of interest to declare.

Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit ( Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

Background Very recently, encouraging results indicate that third party human mesenchymal stromal cells (hMSCs) are a rapidly available therapeutic tool for the treatment of severe (grade III–IV), steroid resistant, acute graft versus host disease (aGVHD). In the clinical experience published so far, hMSCs have been expanded in Fetal Bovine Serum (FBS), which may constitute a problem for its antigenicity and as a possible vehicle of animal pathogens. We have established a highly efficient protocol for the in vitro expansion, under strict GMP compliance, of bone marrow derived hMSCs using human platelets lysate (PL) in place of FBS (Capelli C. et al.: BMT, 2007). In this study, upon Ethical Committee approval and patient’s informed consent, hMSCs were administered on a compassionate basis for the treatment of refractory GVHD. Methods hMSCs were prepared from washouts of bags and filters, left over at the end of the standard filtration procedures of the bone marrow harvests from third party HLA mismatched healthy donors. Cells were grown in the presence of DMEM with 5% PL obtained from the Blood Bank of our Hospitals. In a short period of time (10–33 days), low density seeding of unmanipulated cells (100–200/cm2), obtained from 7 bone marrow harvests allowed to prepare large quantities of hMSCs (median 115×106, range: 67–375), with only one in vitro passage. Twenty-three frozen bags of hMSCs (each containing approximately 1×106/kg of recipient body weight) have been quarantined until the completion of quality tests, including viability, phenotype, absence of detectable bacteria, fungi, mycoplasma or endotoxin, according to European Pharmacopea guidelines. Differentiation to osteogenic and chondrogenic cells as well as the immunosuppressive potential of these cells was confirmed when tested in mixed lymphocyte reaction (MLR). Q banding and clonogenic assays were performed for each batch and never showed abnormalities of karyotype or autonomous growth in vitro. Results Two adult and 4 pediatric patients were treated for aGVHD (grade II–IV) and 2 adults for extensive chronic GVHD (cGVHD) between January and July 2008, using 12 hMSCs bags that had completed quarantine. Before hMSCs, second or third line treatments had been given to patients with aGVHD, including Etanercept (n= 5), Mycophenolate Mofetil (MMF, n= 4) and Extracorporeal Photopheresis (ECP, n= 3), Rituximab (1 patient). Patients with cGVHD were previously treated with ECP and MMF (n= 2), Imatinib (n= 1) and Etanercept (n= 1). Each infusion contained a median dose of 1×106/kg (range, 0.7–1.2×106) hMSCs. For patients with aGVHD, a single infusion was performed in 4 pediatric patients while 1 and 3 infusions were performed in 2 adult patients. The 2 patients with cGVHD received 1 and 4 infusions, respectively. All infusions were very well tolerated with no immediate or late adverse events according to WHO common criteria. Among pediatric patients with aGVHD, 3 complete and 1 partial responses were registered and all patients are alive and in complete hematologic remission. A complete response was observed in 1 adult with grade III cutaneous aGVHD although the patient rapidly relapsed and died of leukemia progression. No response was observed in the other adult patient who died of progressive grade IV gut and liver aGVHD. The 2 adult patients with cGVHD had both a partial response and are alive. Conclusions These data show that large numbers of third party hMSCs can be expanded in vitro with PL containing medium and stored for immediate use in patients with GVHD. Moreover, the clinical results and the toxicity profile confirm those reported with hMSCs expanded in FBS containing media.

Chick myelomonocytic cells transformed by the v-myb oncogene-containing viruses E26 and AMV differ in that the former resemble myeloblasts and express the v-myb-regulated granulocyte-specific mim-1 gene, while the latter resemble monoblasts and are mim-1 negative. We constructed a series of AMV-E26 chimeras and localized the critical differences between these viruses to three point mutations within the second repeat of the v-myb DNA binding domain. These three positions are altered in the v-myb protein of AMV relative to the proteins encoded by c-myb or E26 v-myb. Back mutating AMV v-myb at any of these three sites restored the oncogene's ability to activate the mim-1 gene. Surprisingly, two of these changes led to the transformation, in vitro and in vivo, of cells having a promyelocyte-like phenotype. These results indicate that different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells, probably by regulating unique sets of differentiation-specific genes.

A somatic point mutation in the JAK2 gene (JAK2V617F) is the key pathogenetic lesion of Polycythemia Vera (PV) and Essential Thrombocythemia (ET) and a significant effort is now paid to identify drugs which may be able to interfere with the JAK2V617Fmutated protein. Among others, one potentially interesting drug family is represented by the Histone Deacetylase Inhibitors (HDACi), which may modify the chromatin structure and ultimately the transcription of many genes, the cell cycle progression and the programmed cell death. ITF2357 is a new HDACi (Italfarmaco, Milan, SpA) that shows a potent anti-proliferative and pro-apoptotic activity against acute myeloid leukemia and multiple myeloma cells and little toxicity against normal hematopoietic and mesenchymal stem cells (Golay J et al.: Leukemia 2007). The most common side effects after its administration to normal volounteers and MM patients are represented by thrombocytopenia and gastrointestinal toxicity. These observations prompted us to investigate the inhibitory activity played by ITF2357 on the autonomous proliferation of cells obtained by PV and ET patients carrying the JAK2V617F mutation and to elucidate the mechanism of action of this inhibition. We first investigated the effect of ITF2357 on the clonogenic activity of cell lines carrying or not the JAK2V617F mutation. ITF2357 inhibited colony formation of HEL cells (an erythroleukemia cell line carrying a JAK2V617F homozygous mutation) with an IC50 of about 0.001 μM. In contrast, the doses of drug required to block colony formation by K562, KG1, NB4 and GF-D8 (all negative for the JAK2V617F mutation) were 100–500 fold higher (IC50 ranging from 0.1 to 0.5 μM). Clonogenic assays were then performed using blood mononuclear cells obtained from 4 PV and 7 ET patients, all carrying the JAK2V617F mutation. Either in the presence or absence (EEC assay) of exogenous growth factors, colonies obtained from JAK2V617F mutated progenitor cells were inhibited at much lower doses of ITF2357 (IC50 0.001 μM) as compared to colonies obtained from JAK2 wild type progenitor cells (IC50 0.1–0.25 μM). When single colonies were picked randomly and analyzed by PCR for the presence of wild type or mutated JAK2V617F alleles, a striking reduction of mutated colonies was detected when ITF2357 was added at 0.001 μM and 0.01 μM, confirming that low doses of ITF357 allow the preferential outgrowth of unmutated over mutated colonies from the peripheral blood mononuclear cells of PV patients bearing JAK2V617F. By Western blotting we also showed that ITF2357 treatment for 24 hours, led to virtual disappearance of total and phosphorylated JAK2V617F in HEL cells whereas it did not affect the wild type JAK2 protein in the control K562 cell line, even after 48 hours in the same conditions. Down-modulation of mutated JAK2V617F was accompanied by specific disappearance of p-STAT5 protein. Finally, by Real time PCR analysis of PV cells treated with ITF2357 for 24 hours, we could demonstrate that this drug does not affect JAK2 mRNA but rather it induces a significant decrease of the PRV1 gene, a known JAK2 target. These data suggest that ITF2357 down-modulates the mutated JAK2V617F protein by post-transcriptional mechanisms and that is followed by inhibition of p-STAT5 protein and PRV1 gene expression. The specific inhibition induced by ITF2357 on cells bearing the JAK2V617F mutation underlines its therapeutic potential as a new drug for PV and ET patients.

Rituximab (Mabthera®) is a chimeric monoclonal IgG1 antibody with therapeutic activity in non-Hodgkin B lymphomas (B-NHL) and B-Chronic Lymphocytic Leukemia (B-CLL). We have recently obtained evidence, using a bulky lymphoma xenograft model in nude mice, that both complement and macrophages are required for the therapeutic activity of rituximab. In order to further investigate the tumor cell killing potential of macrophages and its modulation by different factors, including complement, we have set up in vitro experiments with purified macrophage populations. Human macrophages were obtained from purified peripheral blood monocytes cultured for 4 days in presence of 20% FCS and 20 ng/ml M-CSF. FACS analysis confirmed the phenotype of these cells including CD11b and FcγRs expression (CD16, CD32, CD64). Phagocytosis assays were then carried out with CLL cell as targets in presence or absence of increasing concentrations of rituximab. Phagocytosis was evaluated by counting under an inverted microscope the stained cytospin preparations. From 9.8% to 60.8% of macrophages engulfed at least one tumor target cell in a series of 24 experiments (mean 29.7%± 18.3%). Control irrelevant IgG1k monoclonal antibodies (anti-erbB2 trastuzumab and anti-EGFR cetuximab) did not mediate phagocytosis, and rituximab did not lead to ingestion of CD20 negative cells, demonstrating the specificity of the assay. Phagocytosis was already maximal at around 0.1 μg/ml rituximab concentration. In contrast complement activation required Mab concentration of at least 1 μg/ml. Thus phagocytosis, like ADCC, is active at about 10 fold lower MAb concentrations than complement triggering. Levels of CD20 expression on targets did not significantly affect phagocytosis. The role of different FcγRs was also investigated by addition 5 μg/ml blocking antibodies to CD16, CD32 and CD64. All 3 blocking Mabs reduced significantly phagocytosis (by 45%, 42% and 40% respectively with respect to control). Inhibition increased to 64% in presence of all 3 antibodies. Since previous data had suggested a role of the Val/Phe polymorphism at position 158 of CD16A in the clinical response of lymphoma patients to rituximab as well as in NK-mediated ADCC, we investigated whether this polymorphism also affected phagocytosis. No significant differences in dose response curves were observed using macrophages from either Val-Val or Phe-Phe homozygotes. Perhaps surprisingly, concomitant complement activation induced by addition of human serum did not increase phagocytosis. Whether human macrophages can also mediate antibody dependent cellular cytotoxicity (ADCC) was also studied. CLL or BJAB cells were labeled with Calcein-AM and ADCC measured as released fluorescence after 4 hours at 37°C. Macrophages were unable to mediate ADCC in presence of rituximab even following treatment with IFNγ (100 U/ml) for 48 hours. We conclude that macrophages efficiently mediate phagocytosis but not ADCC in presence of low concentrations of rituximab.

Cytokine induced killer cells (CIK) are T/NK cells with demonstrated anti-tumoral activity but lack of GVHD reactivity. They are expanded in vitro after stimulation of PBMC with OKT3, IFN-γ and rhIL-2. This phase I study was designed to test the safety and feasibility of repeated infusions of in vitro expanded donor derived CIK cells given to patients relapsed after allogeneic HSCT. The mean number of starting total nucleated cells was 707 × 106 (range 58–1500 × 106). After a median 22 days of culture, a mean percentage of 51% CD3+CD56+ cells (range 40–71%) was obtained corresponding to an absolute mean number of 1421×106 total CIK cells (range 422–2470 × 106). Eleven patients with AML (n=4), HD (n=3), CMML (n=1), pre-B ALL (n=1) and MDS (n=2), all relapsed after sibling (6) or matched unrelated donor (5) HSCT, entered this study. Before CIK administration, 7 patients had received one or more additional salvage treatments including chemotherapy (5), radiotherapy (1) and unmanipulated DLI (6) without any significant tumor response. The median number of CIK infusions was 2 (range 1–7) and the median number of total CIK cells was 14.5 ×106/kg (7.2–51). The infusions were well tolerated and no acute or late infusion-related reactions were registered. Acute GVHD (grade I and II) was observed in 4 patients 30 days after the last CIK infusion, which progressed into extensive chronic GVHD in 2 cases. In 6 patients, no significant clinical response could be registered so that disease progression and death occurred rapidly. In contrast, 5 patients achieved measurable responses: a patient with MDS, who had been treated with CIK cells alone, showed a hematologic improvement but subsequently progressed and died. One patient with HD received local radiotherapy and 7 CIK infusions (total of 51×106/kg CIK cells) which allowed the achievement of a good PR. After almost 1 year (300 days), he progressed and chemotherapy was given with achievement of a very good PR. A second patient with HD received one DLI at day 516 and 1 CIK infusion (12.2×106/kg) at day 537. At day 572, chemotherapy was initiated due to the persistence of disease. At the end of chemotherapy he received 3 additional CIK infusions for a total of 34×106/kg from days 711–752, without signs of aGVHD and is presently in CR at more than 780 days. One patient with CMML had been treated with DLI on day 102 and with a total of 38×106/kg CIK cells, given in four infusions from days 137–530, because of the appearance of mixed chimerism. Full chimerism was achieved 42 days after the first CIK infusion. The patient remains in CR at day 576. A second MDS patient, who had not achieved any significant response after five DLI (days 411–559), obtained a complete hematologic, cytogenetic and molecular remission after a single CIK infusion (7.6 ×106/kg) given at day 603 and remains in CR at day 680. This study shows that the production of allogeneic CIK cells is feasible, their infusion is generally safe and may induce clinical remission in patients relapsing after HSCT.

Cytokine induced killer cells (CIK) are CD3+/CD56+ T/NK cells with cytotoxic potential against leukemic and other tumor cells but not normal bone marrow in vitro and in vivo. They are expanded in vitro with rhIL-2 after stimulation of peripheral blood mononuclear cells with OKT3 and IFN-γ. We have shown in a recent phase I study that 107/kg allogeneic CIK cells can be safely given to patients relapsing after allogeneic bone marrow transplantation and show evidence of anti-leukemic activity in vivo with very little GVHD. Cord blood (CB) transplantation is progressively becoming an extensively used treatment for patients with malignant disorders. One major limitation of this procedure is the lack of donor derived cells to perform donor lymphocyte infusions in case of relapse. In order to be able therefore to extend the use of CIK cells to the CB transplantation setting, we have standardised a 21 days expansion protocol to produce CIK cells starting from very small amounts of nucleated cells isolated from cord blood. Using this protocol, 15x106 mononuclear cells (MNC) from CB containing a mean 0.3x106 CD3+/CD56+ yielded on average 805 x 106 MNC (50 fold expansion) containing 630 x106 CD3+/CD56+ cells (corresponding to a fold expansion of 1860 for CIK). In order to transfer the method to a clinical setting, we explored the possibility of expanding the residual cells recovered from the empty bags after CB transplantion. Three used CB bags were returned to the laboratory after transplantation and repeatedly washed. An average of 22 x106 nucleated cells could be recovered, yielding a mean 473 x106 CD3/CD56+ cells at the end of the culture period (1485 fold expansion of CIK cells). CIK cells generated from CB showed strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias (42–72% killing at a 30:1 E:T ratio). More importantly, they were cytotoxic against AML blasts isolated from 2 patients (41% lysis). During expansion CB derived CIK cells upregulated the NKG2D marker from a mean fluorescence intensity of 49 to 209. Furthermore they expressed perforin and granzyme molecules in >90% of cells. These observations open up the possibility of a future clinical application of this protocol, performed in GMP conditions. Patients relapsing following cord blood transplantation may be treated with CIK cells expanded from the same cord blood unit, where donors would not be anymore available for cell mediated immunotherapy.

Background In Polycythemia Vera (PV) and Essential Thrombocythemia (ET) hematopoietic progenitor cells can proliferate in vitro in the absence of exogenous growth factors. A somatic point mutation in the JAK2 gene (JAK2V617F) has been recently recognised as the key pathogenetic lesion of these diseases leading to constitutive tyrosine phosphorylation of JAK2, cytokine hypersensitivity and autonomous outgrowth of hematopoietic progenitor cells. Hystone-Deacetylase inhibitors (HDACi) are known inducers of cell differentiation, apoptosis and cell cycle arrest of neoplastic cells. ITF2357 is a new HDACi (Italfarmaco, Milano, Italy) which, at low micromolar concentration in vitro, inhibits the secretion of several cytokines such as IL-1, IL-6, VEGF and IFN-g and exerts a potent anti tumor activity against multiple myeloma (MM) and acute myeloid leukemia cells (AML) (Golay et al., submitted). ITF2357 is well tolerated when given to normal healthy volounteers and Phase II clinical trials are currently ongoing in AML and MM. Aim To investigate the ability of ITF2357 and the prototypic HDAC inhibitor Suberoyl Anilide Hydroxamic Acid (SAHA) used as control, to inhibit the spontaneous outgrowth of hematopoietic stem cells obtained from patients with PV (n= 6, all JAK2V617F ), ET (n= 13, 7 JAK2V617F ) and Idiopathic Erythrocytosis (IE, n= 6, all negative for JAK2V617F ). Results Endogenous erythroid colonies (EEC) assays were performed using mononuclear cells (MNC) from peripheral blood samples obtained from patients at the time of regular follow-up visits in our clinic. MNC obtained from IE or ET patients negative for JAK2V617F neither exhibited spontaneous EEC formation nor Epo hypersensitivity (from 0.1 UI/ml up to 10UI/ml). On the contrary, MNC from JAK2V617F PV and ET patients invariably sustained the spontaneous EEC outgrowth with a marked Epo hypersensitivity. When ITF2357 was added to the colony assay (ranging from 0.001 to 0.75 μM), a 90% inhibition of EEC formation was observed in all JAK2V617F PV and ET patients at 0.01 μM concentration, which corresponds to a blood level easily attained following oral administration of safe doses of ITF2357 to healthy individuals. By contrast, the prototypic HDAC inhibitor SAHA displayed a similar inhibitory activity on EEC formation only when used at 0.25 μM. By flow cytometry experiments performed on mature granulocytes isolated from PV patients we could show that ITF2357 does not modulate the overexpression of Leucocyte Alkaline Phospatase and CD177 (the PRV-1 gene product) thus suggesting that the inhibitory activity on hematopoietic cells is mainly due to a direct action on the stem cell compartment. Conclusion ITF2357, at concentration easily attained after low oral doses of the drug, show a potent inhibitory activity on the autonomous proliferation of hematopoietic stem cells of PV and TE carrying the JAK2 V617F mutation. This may provide the framework for a Phase II study of ITF2357 in these malignancies.

ITF2357 (Italfarmaco, Italia) is a novel hydroxamic acid-based HDAC inhibitor (HDACi) that has shown reduced toxicity in phase I studies. We have investigated the cytotoxic and anti-proliferative activities of ITF2357 in multiple myeloma (MM) and acute myelogenous leukaemia cells (AML). ITF2357 had a strong cytotoxic activity in 8/9 MM and 6/6 AML cell lines, with a mean IC50 of about 0.2 μM, a concentration largely attained following oral administration of safe doses of ITF2357 to healthy individuals. In contrast SAHA, the prototypic hydroxamic HDACi, showed an IC50 of about 1 μM or above in all cases. The cytotoxic activity of ITF2357 was due to induction of apoptosis, as documented by detection of annexin V and cleaved caspase 3. The ITF2357 induced hyperacetylation of histones in cell lines resistant or sensitive to the cytoytoxic activity of the drug is under investigation in order to further define its mechanism of action. ITF2357 had also more potent cytotoxic activity compared to SAHA against freshly isolated CD138+ purified MM cells and AML samples, with an IC50 of about 0.1 μM in 3/3 MM and 13/15 AML cases. Sensitive AML cases included five cases of FAB M1, five M2 and three M4. Three of the sensitive cases carried a t(8;21) translocation, 2 an inv(16), 6 had a normal and 2 a complex karyotype. Furthermore four of the sensitive AML also carried a flt3 internal tandem duplication and 3 a type A mutation in the nucleophosmin 1 gene (NPM1). The 2 more resistant AML cases (one M1 and one M5, both with normal karyotype) showed nonetheless a response to the drug with an IC50 of about 0.5 μM. We have also developed a culture system to grow freshly isolated AML cells for at least 3 weeks in vitro on human mesenchymal cells (MSC). Interestingly, ITF2357 was cytotoxic for primary AML cells stimulated to grow in optimal conditions on MSCs, at the same dose as in standard short term cultures. In contrast ITF2357 was not cytotoxic for MSCs even at the 1 μM concentration. The strong cytotoxic activity of ITF2357 on MM and AML has provided the framework for ongoing phase I studies of ITF2357 in these malignancies.

We have investigated CD52 expression and alemtuzumab (Campath-1H®) mediated lysis in acute lymphoblastic leukaemia (ALL) cell lines and freshly isolated samples. CD52 was expressed in 4 out of 9 ALL cell lines studied (44%), only two of which expressed it at high levels: the preB ALL REH carrying a t(12;21) translocation and the B-ALL Silti with rearranged c-myc (both with MFI of about 270). In contrast, of 57 freshly isolated ALL samples analysed, 86% of cases expressed CD52, albeit at varying levels. CD52 expression was generally more frequent and more intense on more mature ALL along either the B or T lineage. In particular none of the 4 proB-ALL carrying a t(4;11) translocation expressed CD52 (0%), whereas 100% of B linage ALL with a t(12;21), t(9;22) or t(1;19) translocations (total 19 cases analysed) stained positive for CD52 at varying levels (100%). We next analysed alemtuzumab and complement mediated lysis (CDC) and ADCC. ADCC was equivalent in different CD52+ lines, reaching 50% lysis at a 60:1 effector:target ratio. In contrast complement dependent cytotoxicity was variable in different cell lines, since the REH cell line bearing the t(12;21) translocation showed 47% lysis at 10 μg/ml alemtuzumab compared to 0–6% for the other cell lines (Silti and the B lymphoma EsIII), expressing equivalent amounts of CD52. Interestingly amongst freshly isolated ALL, 10/10 t(12;21) ALL samples showed very high CDC (mean 96% lysis) in presence of 10 μg/ml alemtuzumab and human serum, compared to the other 15 CD52+ cases tested in the same conditions which showed a mean of 19% lysis (p

Eight patients with advanced ovarian carcinomas resistant to conventional chemotherapy were injected with interferon (IFN) beta (3 X 10(6) U) intraperitoneally twice a week. Seven subjects had ascites. Side effects included abdominal pain, fever, and constipation, but no hematologic toxicity was observed. Growth of solid tumor lesions was unaffected by IFN beta, with the possible exception of one patient who had stable disease. IFN beta intraperitoneally inhibited completely the formation of ascites in four of seven patients with effusions. Natural killer (NK) cell activity was measured in peripheral blood and tumor-associated lymphocytes (PBL and TAL). Using stringent criteria that included repeated assessment of baseline activity, a clear cut increase in NK cytotoxicity of TAL was detected in two of six subjects from whom TAL could be purified. Augmentation of NK activity was restricted to the peritoneal compartment with no effect on PBL. Studies on biologic response modifiers encompassing an analysis of events taking place at sites directly involved by neoplasia may provide an opportunity for generating information on the in situ regulation of tumor-associated host defense mechanisms in humans.

Persistent unexplained lymphadenopathy (LAS) with intermittent fever, weight loss, night sweats and malaise was observed from March to October 1983 in 16 of 133 intravenous drug addicts who had been followed for at least two years in a Center for Drug Addicts Assistance in Milan, Italy. All the subjects lived in a restricted suburban area and indulged in frequent toxicomanic practices and mutual sexual intercourse. The subjects showed immunological alterations such as lymphopenia (50%), decreased T helper/T suppressor ratio (93%), both these abnormalities (43%), decreased T helper cells (75%), increased T suppressor cytotoxic cells (81%), decreased natural killer (NK) activity (77%), anergy (50%) or hypoergy (43%) to recall skin testing and elevated levels of IgG (87%). Anti-HTLV III antibodies were found in 14 of 16 (87%) patients with LAS and in 3 of 11 (27%) symptom-free drug addicts belonging to the same group. It will be important to assess in the future whether this clinical and immunological picture results in acquired immunodeficiency syndrome in an area so far untouched by this disease.

The streptococcal preparation OK432 was studied for its effects on natural killer (NK) activity of peripheral blood lymphocytes (PBL) from normal donors and from ovarian cancer patients, and of tumour-associated lymphocytes (TAL) from peritoneal effusions. OK432 augmented NK activity against the susceptible K562 line and induced killing of the relatively resistant Raji line. Freshly isolated ovarian carcinoma cells were relatively resistant to killing by unstimulated PBL and TAL. OK432 induced significant, though low, levels of cytotoxicity against 51Cr-labelled ovarian carcinoma cells. Augmentation of killing of fresh tumour cells by OK432 was best observed in a 20 h assay and both autologous and allogeneic targets were lysed. PBL were separated on discontinuous Percoll gradients. Unstimulated and OK432-boosted activity were enriched in the lower density fractions where large granular lymphocytes (LGL) and activity against K562 were found. Thus, OK432 augments NK activity of PBL and TAL in human ovarian carcinomas and induces low, but significant, levels of killing of fresh tumour cells. Effector cells involved in killing of fresh ovarian tumours copurify with LGL on discontinuous gradients of Percoll.

The v- myb , ets -containing avian leukemia virus E26 is unique in its capacity to transform both erythroblasts and myeloblasts. Previous studies showing that v- myb is sufficient for the transformation of myeloid cells failed to definitively establish the role of the v- ets gene. We have now isolated a mutant of E26, ts 1.1, that is temperature-sensitive for erythroid cell transformation and that we found to contain a single mutation in the v- ets gene. Surprisingly, myeloid cells transformed by this mutant showed an altered phenotype relative to wild-type-transformed cells, in that they resemble promyelocytes. In addition, infection of mature macrophages with ts 1.1 led to their transformation and conversion into promyelocyte-like cells. We conclude that the v- ets domain of the p135 gag-myb-ets protein of E26 has an effect on both erythroid and myeloid cell differentiation, suggesting a possible role for the c-etsc-myb genes in the commitment of hematopoietic cells towards specific lineages.

We have studied 7 patients with T gamma-lymphoproliferative disorders, in whom 78-88% of circulating nonadherent lymphocytes had the morphology of large granular lymphocytes (LGL) as assessed by light and transmission electron microscopy. The main common features of the membrane phenotype of these LGL expansions included expression of T3, HNK-1 and AB8.28. Other monoclonal antibody-defined surface markers of LGL (OKM1, B73.1, N901) were variably expressed or absent in these patients. Patients' LGL had little or no natural killer (NK) activity but mediated antibody-dependent cellular cytotoxicity (ADCC). Exposure to interferons (type B or gamma) for 20-72 hr resulted in no appreciable induction of cytolytic activity. In contrast, culture in the presence of interleukin-2 (IL-2) for 3 days resulted in the expression of strong cytolytic activity in all the patients tested against an NK-susceptible (K562) and an NK-resistant (Daudi) target. The expression of T3 antigen, the low levels or lack of native NK activity and the induction of consistent cytotoxicity by prolonged exposure to IL-2 led us to suggest that the cells expanding in these subjects are related to the effectors involved in lymphokine-activated killer (LAK) activity.

C-myb and B-myb belong to the myb family of transcription factors. We have shown previously that c-myb is deregulated in fibroblasts from systemic sclerosis (scleroderma) patients relative to normal fibroblasts. Scleroderma fibroblasts are known to express elevated levels of collagen genes and transforming growth factor beta is known to be a pro-fibrotic cytokine and to induce transcription of type I collagen genes. We have therefore investigated the role of c-myb and B-myb in the regulation of type I collagen genes in response to transforming growth factor beta in normal human fibroblasts. We show that, in these cells, transforming growth factor beta treatment induces c-myb as well as collagen alpha1(I) and alpha2(I) gene expression, but not B-myb. Furthermore we demonstrate by cotransfection assays that c-myb can upregulate alpha1(I) and alpha2(I) collagen promoters by 6-10-fold whereas B-myb is inactive. The activity of c-myb on both type I collagen promoters requires a functional c-myb DNA binding domain suggesting a direct interaction between c-myb and these promoters. Indeed c-myb is active also on a 500 bp fragment of the alpha2(I) collagen promoter and can bind to this fragment in electrophoretic mobility shift assays. Finally, we show that anti-c-myb anti-sense treatment reduces alpha1(I) and to a lesser extent alpha2(I) collagen gene expression. These data strongly suggest that c-myb, but not B-myb, plays a direct role in the upregulation of type I collagen gene expression in response to transforming growth factor beta.

Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix constituents. Although it has been proposed that tissue fibrosis is due to increased fibroblast synthesis of various collagen polypeptides, there is some experimental evidence that patients with systemic sclerosis have a defect in the control of fibroblast growth. The myb family of genes includes, among others, the c-myb proto-oncogene and the structurally related gene, B-myb, which are both implicated in the regulation of differentiation and/or proliferation of hematopoietic and nonhematopoietic cells. To elucidate the molecular basis responsible for scleroderma fibroblast proliferation, we therefore elected to investigate the expression of c-myb and B-myb genes in scleroderma and control cells. Using the reverse transcriptase polymerase chain reaction technique, we detected c-myb transcripts in scleroderma skin fibroblasts rendered quiescent by serum deprivation. Under the same experimental conditions, c-myb message was not found in normal skin fibroblasts, but, after serum stimulation, c-myb RNA was clearly evident from 3 to 72 h in both normal and pathologic cells. Treatment of these cells with c-myb antisense oligonucleotides caused downregulation of c-myb expression, and the inhibition of scleroderma fibroblast proliferation was 42%, whereas in normal fibroblasts the inhibition was weaker (22%). In contrast to c-myb, in normal and scleroderma fibroblasts the level of expression of B-myb correlated with cell proliferation assessed by cell count, and densitometric analysis showed that B-myb message was 1.5-5 times higher in most of pathologic cells studied. The antisense B-myb oligonucleotides had a weaker antiproliferative effect compared with antisense c-myb, inhibiting scleroderma and normal fibroblasts by 23% and 13%, respectively. These data suggest that the B-myb and c-myb genes may play a role in scleroderma fibroblast proliferation and function.

Five patients with aggressive acute leukemias who had relapsed after cord blood transplantation were treated with cord blood derived cytokine-induced killer (CIK) cells. These were obtained by ex vivo expansion, using as starting material the washouts of the cord blood units, left over at the end of the transplant. We did not observe any acute or delayed adverse event, and observed 1 partial response in 1 patient concomitantly with the development of acute grade III graft-versus-host disease (GVHD). These observations show the relatively low toxicity of cord blood-derived CIK cells and, more importantly, the feasibility of this immunotherapy program for patients who could not otherwise benefit from donor lymphocyte infusions. © 2010 American Society for Blood and Marrow Transplantation.

Publisher Summary This chapter examines the natural killer (NK) activity in human ovarian tumors. The defective NK activity of tumor-associated lymphoid cells (TAL) could, in principle, be accounted for by at least two mechanisms, one being the presence of suppressor cells or factors and the other being a low number of the relevant effectors, identified in blood as large granular lymphocytes (LGL), which enter the tumor. In a study described in the chapter, preliminary evidence for the presence in situ of cells inhibiting NK activity was obtained in 3 peritoneal effusions from ovarian cancer in 2 breast carcinomas and in pleural carcinomatous effusions. A relatively large number of ovarian cancer patients are examined for the presence of suppressor TAL or TAM. In part of the experiments, TAL or tumor-associated macrophages (TAM) were mixed in varying ratios with PBL and lysis of K562 cells was measured immediately, whereas in others, suppressors were allowed to interact with PBL for 20 h before adding indicator PBL.TAM were devoid of suppressive activity on NK cells when the assay was performed immediately after mixing, whereas in a minority of TAL preparations, the preliminary indications for the presence of inhibitory cells at the tumor site were confirmed.

Eight ovarian cancer patients with ascites resistant to various chemotherapy protocols were treated i.p. with C. parvum in an effort to obtain a palliative reduction of peritoneal effusions. C. parvum(7–14 mg) was given i.p. on days 0, 7, 28 and then at monthly intervals. Three patients showed no clinical evidence of therapeutic benefit from C. parvum administration. Three subjects had a complete disappearance of ascites, and two additional patients showed a marked reduction of their effusions. Solid tumor masses were not affected by i.p. C. parvum. C. parvum injection caused a rapid decrease of the percentage of tumor cells in the ascites as early as 7–15 days after the first treatment. Concomitantly, the percentage of polymorphs and, to a lesser extent, of macrophages in the ascitic tumor increased, whereas tumor-associated lymphoid cells (TAL) were not altered in a consistent pattern. The tumoricidal activity of peripheral blood monocytes and natural killer (NK) cells was not consistently modified after C. parvum treatment. Pretreatment NK activity of TAL was usually very low and was not appreciably modified by i.p. C. parvum. In one patient, whose TAL had NK activity in the range of normal PBL, C. parvum administration was followed by a profound depression of intratumor NK cytotoxicity. The tumoricidal activity of tumor-associated macrophages (TAM) was not enhanced following C. parvum administration; 14–21 days after the first C. parvum inoculation, a marked decrease of the cytolytic capacity of TAM was observed, cytotoxicity returning to pretreatment values thereafter. These observations do not elucidate the mechanism of the limited, but significant, anti-tumor activity of i.p. C. parvum in human ovarian carcinomatous ascites. The lack of enhancement of macrophage cytotoxicity after i.p. C. parvum, predicted on the basis of data in rodents, cautions against the empirical use in humans of agents assumed to affect human neoplasia through activation of host defense mechanisms.

A study was done to investigate the tumoricidal activity of peripheral blood lymphocytes (PBL) and tumor-associated lymphoid cells (TAL) against freshly isolated tumor cells in human ovarian carcinoma. TAL and carcinoma cells were purified by density and velocity sedimentation on discontinuous Ficoll-Hypaque gradients and fetal bovine serum. Purified carcinoma cells from 23 ascitic and 3 solid tumors were used as targets in a 4- or a 20-hour 51Cr release assay. K562 cells were used to measure natural killer (NK) activity. Freshly purified ovarian carcinoma cells were relatively resistant to lysis by normal unstimulated PBL. Cytolytic activity of ovarian cancer PBL and TAL did not exceed that of control PBL: Only one PBL preparation had high levels of cytotoxicity (36.2 and 42.9% specific lysis after 4 and 20 hr at an effector-to-target cell ratio of 50:1) against autologous cancer cells but not against allogeneic targets (less than 5% specific lysis). TAL and, to a lesser extent, ovarian cancer PBL showed impaired NK activity against K562 compared to the activity seen in controls. In vitro exposure to partially purified human fibroblast interferon (IFN) (1,000 U/ml for 1-18 hr) augmented NK activity against K562 of PBL and TAL. When ovarian carcinoma cells were used as targets, IFN enhanced the cytotoxicity of normal PBL in a 4- and 20-hour assay; stimulation by IFN was less frequently observed with ovarian cancer PBL and TAL in a 4-hour assay, but after 20 hours effector cells from tumor-bearing subjects showed IFN-boosted cytotoxicity against carcinoma cells similar to the degree of cytotoxicity seen in controls. IFN enhanced the cytotoxicity of PBL and TAL against both autologous and allogeneic carcinoma cells. Thus PBL and TAL are rarely cytotoxic against 51Cr-labeled fresh autologous carcinoma cells, but in vitro exposure to IFN induced low levels of killing of ovarian tumor cells.

6 patients with a chronic, clinically heterogeneous proliferative disorder of the large granular lymphocytes (LGL) were investigated. In each case the majority of peripheral blood lymphocytes reacted with HNK-1, OKT3 and T11 monoclonal antibodies, whereas morphology and other immunological features varied from case to case. 2 cases were of particular interest. 1 patient had an expansion of HNK-1 stained, large agranular rather than granular lymphocytes; another patient's LGL simultaneously expressed HNK-1, OKT4 and T8 antigens. The heterogeneous features of these abnormally expanded cell populations are similar to those of the normal cell subsets from which they are likely to have originated.

Natural killer (NK) cells have been studied in human neoplastic diseases in an effort to assess the role of these cells in the control of human neoplasia and to monitor the effects of therapeutic regimens expected to affect this reactivity. NK activity measured against susceptible cell lines is usually somewhat depressed in patients bearing advanced solid tumors, but not at early disease stages. Lymphoid cells associated with solid tumor tissues or effusions have usually low NK cytotoxicity, with considerable differences among histologic types (e.g., nasopharyngeal carcinoma versus other tumors) or at different sites involved by the same tumor (e.g., peritoneal effusions versus solid lesions in ovarian carcinoma). The low levels of NK activity of tumor-associated lymphoid cells are primarily related to a low frequency in the relevant effector cells at the tumor site, although suppression of the in vitro maintenance of cytotoxicity by in situ macrophages and lymphocytes has been described in a few patients. Treatment with immunopharmacologic agents, interferons in particular, has been reported to augment NK activity in cancer patients, but it is unclear how blood NK activity relates to tissue levels of this reactivity. Limited evidence indicates that blood NK levels need not be representative of the activity of tumor associated lymphoid cells. Most studies on NK cells in human neoplasia have dealt with reactivity against susceptible tissue culture lines, but freshly isolated human tumors are generally relatively resistant to these effector cells, particularly when autologous lymphoid cells are used. The resistance of fresh human neoplastic cells to NK activity has not been studied extensively and, together with the poor localization at the tumor site of NK effectors, it represents a major difficulty in envisaging a role for these cells in the control of established human neoplasia.

Cells of the monocyte-macrophage series are a major component of the lymphoreticular infiltrate of rodent and human tumors (1–8). Several studies have focused on the in vitro cytotoxicity of rodent mononuclear phagocytes on tumor cells and on the functional status of tumor-associated macrophages (TAM) from murine neoplasms (3, 9–21). In contrast, little attention has been given to the interaction of human monocytes and macrophages with neoplastic cells, and. Human TAM have not been characterized. Here we will summarize results on the tumoricidal activity of human mononuclear phagocytes and we will discuss issues of current interest in our laboratory.

Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a transient increase in c-fos proto-oncogene mRNA levels (Introna et al., 1986). After 2 h from the initial treatment, c-fos mRNA could no longer be detected and its expression could not be restimulated either by LPS or by other signals including colony stimulating factor-1 (CSF-1) and phorbol myristate acetate (PMA), both of which are able to induce expression of the c-fos gene in unstimulated macrophages. When LPS was removed after an initial 30 min incubation, responsiveness to a second exposure to LPS began to reappear after 3 h and was completely restored by 20 h. The same pattern of desensitization of c-fos induction was observed when CSF-1 stimulated macrophages were subsequently exposed to LPS. The loss of sensitivity to PMA following pretreatment with LPS was selective for c-fos expression as LPS treated macrophages remained responsive to PMA with respect to the ability to stimulate secretion of H2O2. The mechanism of desensitization was localized, at least in part, at the level of transcription as demonstrated by analysis of c-fos transcripts in nuclei isolated from macrophages pretreated and restimulated with LPS.

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide (LPS) has been previously documented to induce accumulation of mRNA for the early or competence genes JE and KC; the data further suggested that multiple pathways existed for the transduction of the LPS signal, since induction of mRNA for JE was related to breakdown of polyphosphoinositides while induction of KC was not (Introna et al. 1987 J. Immunol. 138, 3891). This study provides analysis of the regulation of the expression of these genes by using the nuclear transcription assay. We present evidence that LPS enhanced transcriptional activity of the KC gene, but not of the JE gene. By contrast, serum stimulation of quiescent BALB c -3T3 fibroblasts induced transcription of the JE and KC genes. The data imply that expression of the KC gene in LPS-treated macrophages is regulated transcriptionally, while that of the JE gene is regulated post-transcriptionally. Furthermore, there appear to be two mechanistic pathways for the induction of JE mRNA depending upon the stimulus and upon the cell type: one involving transcriptional and one post-transcriptional control.

Tumor-associated lymphocytes (TAL) were isolated from 17 ascites and 7 solid ovarian carcinomas. TAL had defective natural killer (NK) activity against K562. Large granular lymphocytes, the morphologically identified effectors of NK activity, were poorly represented in TAL from ovarian carcinomas as compared to peripheral blood lymphocytes from the same patients or from normal donors. Similar results were obtained when effectors of NK activity were identified with an anti-NK (HNK-1) monoclonal antibody. When four TAL preparations were separated on discontinuous Percoll gradients, they were able to be enriched for NK activity and large granular lymphocyte morphology in the lower density fractions as observed with blood. These observations suggested that a low concentration of the relevant effector cells was the major factor determining the defective NK cytotoxicity of lymphoid cells associated with these human neoplasms.