CIK as therapeutic agents against tumors.

Cytokine Induced Killer (CIK) cells are ex vivo expanded and activated T lymphocytes obtained by sequential incubation of Peripheral Blood Mononuclear cells (PBMNC) with Interferon γ (IFNG), anti CD3 monoclonal antibody OKT3 and IL2. These cells, while retaining few characteristics of the Effector memory T cells subpopulation, acquired during culture CD56 expression, as well as non specific, Natural Killer like, anti tumoral cytotoxicity. CIK cells from human are equivalent to expanded NKT cells in mouse. More interestingly, CIK cells show a potent intratumoral homing in several experimental models, followed by anti tumoral clinical activity in mice and humans. In spite of extensive in vivo permanence and proliferation, CIK cells do not show cytotoxicity against normal targets and, particularly important, do not show Graft versus host disease when tested in allogeneic combinations (donor versus host) even in the haploidentical matching. For the easiness of the laboratory preparations, the availability of clinical grade reagents, the production of Good Manufacturing Practice compliant methods, CIK cells have been extensively used for the treatment of cancer patients, in both hematologic and solid tumors, in both autologous and allogeneic combinations. Several clinical protocol will be here discussed and summarised to show the feasibility of these passive transfer approaches, and also their very limited toxicity. Finally, preliminary indications on clinical efficacy, particularly in hematologic malignancies and against minimal residual disease, will be shown and discussed, as well as the future perspectives to optimize this adoptive passive cell immunotherapy strategy by gene transfer technology or bispecific monoclonal antibodies addition.

Clinical grade expansion of MSCs.

Producing advanced therapy medicinal products (ATMP) according to Good Manufacturing Practice (GMP) guidelines represents a global challenge for the expansion of cells intended for human use. Mesenchymal stromal cells (MSCs) from different sources are one of the most actively developed cell type for a variety of clinical applications in cellular therapy. Complying with GMP means defining accurately both the production process and the release criteria required for a final safe product. We have here reported our manufacturing experience on 103 consecutive clinical-grade in vitro expansions of both bone marrow-derived and umbilical cord-derived mesenchymal stromal cells together with description of methods and reagents utilized in our Cell Factory. The same animal- and serum-free medium, additioned with human platelet lysate, has been used for all the expansions performed. This is the largest experience published so far with this alternative and clinical-grade reagent (compared to the traditional fetal bovine serum) and shows the feasibility and the reproducibility of the method. Indeed, we have been able to produce a sufficient number of MSCs to treat 57 patients so far, enrolled in 7 different experimental phase I/II protocols.

Cell-based strategies to manage leukemia relapse: efficacy and feasibility of immunotherapy approaches.

When treatment fails, the clinical outcome of acute leukemia patients is usually very poor, particularly when failure occurs after transplantation. A second allogeneic stem cell transplant could be envisaged as an effective and feasible salvage option in younger patients having a late relapse and an available donor. Unmanipulated or minimally manipulated donor T cells may also be effective in a minority of patients but the main limit remains the induction of severe graft-versus-host disease. This clinical complication has brought about a huge research effort that led to the development of leukemia-specific T-cell therapy aiming at the direct recognition of leukemia-specific rather than minor histocompatibility antigens. Despite a great scientific interest, the clinical feasibility of such an approach has proven to be quite problematic. To overcome this limitation, more research has moved toward the choice of targeting commonly expressed hematopoietic specific antigens by the genetic modification of unselected T cells. The best example of this is represented by the anti-CD19 chimeric antigen receptor (CD19.CAR) T cells. As a possible alternative to the genetic manipulation of unselected T cells, specific T-cell subpopulations with in vivo favorable homing and long-term survival properties have been genetically modified by CAR molecules. Finally, the use of naturally cytotoxic effector cells such as natural killer and cytokine-induced killer cells has been proposed in several clinical trials. The clinical development of these latter cells could also be further expanded by additional genetic modifications using the CAR technology.

Influence of substituting dietary soybean for air-classified sunflower (Helianthus annuus L.) meal on egg production and steroid hormones in early-phase laying hens.

Soybean meal (SBM) is the most widely and expensive protein source used in the formulation of poultry diets; however, when the price of SBM increases, poultry nutritionists seek alternative sources that are more economical in formulating least-cost rations. This research aimed to evaluate the effects of dietary air-classified sunflower meal (SFM) on some productive parameters and plasma steroid hormones in laying hens. In this trial, 20-week-old laying hens (ISA Brown strain) in the early phase of production were randomly assigned to two groups and fed wheat middlings-based diets containing soybean (135 g/kg; 48% CP) or air-classified SFM (160 g/kg; 41% CP) as the main protein source. Laying performance, egg size and feed conversion ratio were evaluated for 10 week. Plasma steroid hormones (progesterone and oestradiol) in the hens were quantified weekly. Substituting SBM with air-classified SFM did not change (p > 0.05) the hens' growth performance, whereas feed consumption and efficiency were positively influenced (p < 0.05) by SFM treatment. Egg production rate was improved in hens fed the SFM diet (p < 0.05), as well as the percentage of medium-size eggs that was higher for SFM treatment (p < 0.05). Steroid hormones levels were affected by dietary treatment (p < 0.01). From our findings, it could be effective to include air-classified SFM in early-phase laying hen diets as an alternative protein source substituting SBM, without negative influence on productive performance and egg traits, reducing also the production costs.

Cytokine Induced Killer (CIK) cells for the treatment of haematological neoplasms.

Cytokine Induced Killer (CIK) cells are in vitro activated human CD8 T cells which have maintained several characteristics of T-EMRA cells and additionally acquired non specific anti tumoral cytotoxicity and CD56 overexpression, thus representing a cell population with double T and NK phenotype. Due to their in vivo intratumoral homing and lack of Graft versus Host (GVH) reactivity, CIK cells have been extensively used in cancer patients either in autologous or allogeneic contexts. Here we summarise CIK main biological features as well as their most prominent clinical results.

Localization of mesenchymal stromal cells dictates their immune or proinflammatory effects in kidney transplantation.

Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell-based immunotherapy in solid-organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg-dependent mechanism. MSC-infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation.

The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).

We investigated the activity of ITF2357, a novel histone deacetylase inhibitor (HDACi) with antitumor activity, on cells carrying the JAK2(V617F) mutation obtained from polycythemia vera (PV) and essential thrombocythemia (ET) patients as well as the HEL cell line. The clonogenic activity of JAK2(V617F) mutated cells was inhibited by low concentrations of ITF2357 (IC(50) 0.001-0.01 microM), 100- to 250-fold lower than required to inhibit growth of normal or tumor cells lacking this mutation. Under these conditions, ITF2357 allowed a seven fold increase in the outgrowth of unmutated over mutated colonies. By western blotting we showed that in HEL cells, ITF2357 led to the disappearance of total and phosphorylated JAK2(V617F) as well as pSTAT5 and pSTAT3, but it did not affect the wild-type JAK2 or STAT proteins in the control K562 cell line. By real-time PCR, we showed that, upon exposure to ITF2357, JAK2(V617F) mRNA was not modified in granulocytes from PV patients while the expression of the PRV-1 gene, a known target of JAK2, was rapidly downmodulated. Altogether, the data presented suggest that ITF2357 inhibits proliferation of cells bearing the JAK2(V617F) mutation through a specific downmodulation of the JAK2(V617F) protein and inhibition of its downstream signaling.

Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts.

We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P=0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 x 10(6) to 365 x 10(6) hMSCs in PL versus a variable number from 2.7 x 10(6) to 31 x 10(6) in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.

The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.

We have used a standardized 21-day expansion protocol to produce cytokine-induced killer (CIK) cells starting from very small amounts of nucleated cells (approximately 15 x 10(6) cells) isolated from cord blood. Mononuclear cells are stimulated with anti CD3 (OKT3) and IFNgamma and then expanded with IL-2. Moreover, we show that washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500 x 10(6) CIK by the same expansion protocol. CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels. The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions and therefore these observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.

Gemtuzumab ozogamicin (Mylotarg) has therapeutic activity against CD33 acute lymphoblastic leukaemias in vitro and in vivo.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated with the cytotoxic drug calicheamicin and approved for the treatment of relapsed acute myeloid leukaemia. As approximately 18% of acute lymphoblastic leukaemias (ALL) are also CD33 positive, we have investigated the cytotoxic activity of GO on CD33+ ALL cells in vitro and in vivo. 10 ng/ml GO induced 30-95% inhibition of thymidine uptake and 30-70% cell death in four freshly isolated and one in vivo passaged CD33+ ALL-cell cultures. Furthermore, an in vivo model of a CD33+ ALL carrying the Philadelphia chromosome [t(9;22)] was established. 5 x 10(6) ALL-2 cells inoculated in the tail vein of severe combined immunodeficient mice engrafted into haematopoietic organs, reaching a mean of 70%, 61% and 69% human CD45+ cells in bone marrow, spleen and liver, respectively, at 35 d. To test the therapeutic activity of GO, 50 or 100 microg immunotoxin was inoculated i.p. on days 7, 11 and 15 following tumour-cell inoculation. GO treatment dramatically inhibited expansion of ALL-2 cells in all tested organs and increased survival of tumour-injected animals by 28-41 d, relative to controls. These data demonstrated that GO is active both in vitro and in vivo against CD33+ ALL cells.

Scleroderma fibroblasts constitutively express the long pentraxin PTX3.

OBJECTIVE: PTX3 is a secreted molecule which consists of a C-terminal domain similar to classical pentraxins (e.g. C-reactive protein) and of an unrelated N-terminal domain. Unlike the classical pentraxins, PTX3 is expressed in response to IL-1beta and TNF-alpha but not to IL-6. The present study was designed to investigate the expression of PTX3 in normal and scleroderma fibroblasts. METHODS: Normal and SSc fibroblasts were cultured in the presence and absence of inflammatory cytokines. PTX3 mRNA expression in fibroblasts was evaluated by Northern analysis. PTX3 protein levels in fibroblast culture medium were estimated by ELISA. RESULTS: Normal fibroblasts were induced to express high levels of P7X3 mRNA by IL-1beta and TNF-alpha but not by other cytokines or growth factors. Scleroderma fibroblasts, unlike normal fibroblasts, constitutively expressed high levels of PTX3 in the absence of deliberate stimulation. The constitutive expression of PTX3 in SSc fibroblasts was not modified by anti-TNF-alpha antibodies or IL-1 receptor antagonist. In contrast, IFN-gamma and TGF-beta inhibited the constitutive but not the stimulated expression of PTX3 in SSc fibroblasts. CONCLUSIONS: PTX3 is a main feature of activated scleroderma fibroblasts.

Modulation of cell cycle by graded expression of MLL-AF4 fusion oncoprotein.

Acute lymphoblastic leukemia (ALLs) expressing MLL-AF4, the fusion product of t(4;11)(q21;q23), show marked leucocytosis and extramedullary disease in multiple organs, respond poorly to chemotherapy and have poor prognosis. In vitro, leukemic cells with the t(4;11) show resistance to serum deprivation-induced or interferon gamma-regulated CD95-mediated apoptosis. In addition, t(4;11) cells have prolonged doubling time and lower percentage of cells in cycle compared to non-t(4;11) B lineage cell lines. In this study, we examine the time- and level-dependent effects of MLL-AF4 conditional expression on cell cycle and differentiation of myelomonocytic leukemia cell line U937. By varying the concentration of tetracycline in growth media, we found that increasing levels of MLL-AF4 expression result in a progressive decrease in growth rate and fraction of S phase cells, paralleled by an increase in percentage of cells expressing CD11b. Our results demonstrate a dosage-dependent effect of MLL-AF4 fusion oncoprotein on cell cycle progression, with increasing expression levels resulting in the accumulation in G1, prolonged doubling time, both findings that might be responsible for the increased resistance to etoposide-mediated cytotoxicity. We propose the cell cycle control exerted by MLL-AF4 may be responsible of resistance to cell-death promoting stimuli in leukemia carrying the t(4;11) translocation.

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors.

Three different second-generation lentiviral self-inactivating vectors containing CMV, EF1alpha and PGK promoter, respectively, and all carrying the exogenous GFP gene, were compared for expression in human B-cell precursor ALL blasts. At a comparable percentage of transduction and vector DNA copy number, CMV clearly showed better efficiency of transcription. Human bone marrow stromal cells were favored compared to the MRC-5 cell line, as support for cell viability during infection. Cells were infected and analyzed after variable culture times ranging from 4 to 12 days, to reduce the possibility of pseudotransduction. In 10/14 samples, we detected more than 20% GFP-positive cells after exposure to high-titer viral supernatants. We then tested a similar vector carrying the human CD40L cDNA and, in similar infection conditions, obtained more than 20% transduction in 6/6 samples. The levels of transduction obtained were sufficient to induce the upregulation of CD83 molecule in cocultured immature dendritic cells.

CD40 ligand-stimulated B cell precursor leukemic cells elicit interferon-gamma production by autologous bone marrow T cells in childhood acute lymphoblastic leukemia.

Childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, collected from bone marrow (BM) at diagnosis, were cultured, after thawing, on allogeneic human bone marrow stroma (HBMS) for 48 h in the presence of a soluble trimeric CD40 ligand (stCD40L) molecule. HBMS maintained leukemic cells viability in all tested cases (mean viability 85%). Under these culture conditions we noticed upregulation or de novo expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) in 22/22, 15/23 and 21/23 cases, respectively. Upregulation, in terms of fluorescence intensity, was also observed in the expression of MHC I, MHC II, CD54 (ICAM 1) and CD58 (LFA 3) molecules. HBMS alone, although to a lesser extent, was able to induce modulation of these molecules, but not CD80, in a similar proportion of cases. Neither stCD40L nor HBMS induced modulation of CD10 and CD34 molecules. Moreover, in 4/4 tested cases, stCD40L-stimulated ALL cells were able to induce allogeneic T cells proliferation. To evaluate whether leukemia-reactive T cells were detectable in the BM of ALL patients at diagnosis, stCD40L-stimulated ALL cells were co-cultured with autologous T cells (ratio 1:1), isolated from BM at diagnosis, for 4 days and a 24 h ELISPOT assay was applied to detect the presence of interferon-gamma (IFN-gamma)-producing cells. In four of seven cases IFN-gamma-producing cells were detected with frequencies of 1/900, 1/1560, 1/2150 and 1/1575 autologous T cells. These data confirm that stCD40L exposure can activate the antigen-presenting cell (APC) capacity of BCP-ALL cells cultured on HBMS and that ELISPOT assay can be used to measure the frequency of leukemia-reactive autologous T cells in the BM of ALL patients even after short-term culture with stCD40L-stimulated ALL cells.

CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59.

Complement-dependent cytotoxicity is thought to be an important mechanism of action of the anti-CD20 monoclonal antibody rituximab. This study investigates the sensitivity of freshly isolated cells obtained from 33 patients with B-cell chronic lymphocytic leukemia (B-CLL), 5 patients with prolymphocytic leukemia (PLL), and 6 patients with mantle cell lymphoma (MCL) to be lysed by rituximab and complement in vitro. The results showed that in B-CLL and PLL, the levels of CD20, measured by standard immunofluorescence or using calibrated beads, correlated linearly with the lytic response (coefficient greater than or equal to 0.9; P <.0001). Furthermore, the correlation remained highly significant when the 6 patients with MCL were included in the analysis (coefficient 0.91; P <.0001), which suggests that CD20 levels primarily determine lysis regardless of diagnostic group. The role of the complement inhibitors CD46, CD55, and CD59 was also investigated. All B-CLL and PLL cells expressed these molecules, but at different levels. CD46 was relatively weak on all samples (mean fluorescence intensity less than 100), whereas CD55 and CD59 showed variability of expression (mean fluorescence intensity 20-1200 and 20-250, respectively). Although CD55 and CD59 levels did not permit prediction of complement susceptibility, the functional block of these inhibitors demonstrated that they play an important role in regulating complement-dependent cytotoxicity. Thus, lysis of poorly responding B-CLL samples was increased 5- to 6-fold after blocking both CD55 and CD59, whereas that of high responders was essentially complete in the presence of a single blocking antibody. These data demonstrate that CD20, CD55, and CD59 are important factors determining the in vitro response to rituximab and complement and indicate potential strategies to improve the clinical response to this biologic therapy.

Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug alone.

We have shown previously that the anti-CD20 chimaeric monoclonal antibody rituximab exerts its effects on neoplastic B-lymphoma cell lines in part via complement-dependent cytotoxicity. In addition, membrane expression levels of complement inhibitory proteins CD55 and CD59 play a role in determining susceptibility to lysis. We have identified one t(14;18)-positive human B-cell non Hodgkin's lymphoma cell line (Karpas 422) that is resistant to rituximab and complement and used it for subsequent studies on the possible interaction between this novel therapeutic agent and established antineoplastic drugs. We have exposed Karpas to several chemotherapeutic agents (doxorubicin, idarubicin, cisplatin, taxol) for different time periods and subsequently exposed the cells to rituximab and human complement. The combination of these drugs with rituximab induced an additive cytotoxic effect. In contrast, exposure to fludarabine (1 microg/ml for 48-72 h) showed a synergistic effect, with cell lysis increasing from 10% to 20% using fludarabine or rituximab and complement alone to about 70% with both cytotoxic agents. Analysis of the mechanism for this synergistic effect showed that fludarabine downmodulates the membrane expression of CD55 (from 96% to 55% positive cells) without significantly altering CD20 levels. Northern analysis demonstrated that fludarabine induced a general downmodulation of steady state mRNA levels with no change in transcription rate detected in run-off assays. The study of the effect of fludarabine and rituximab in six freshly isolated B-cell chronic lymphocytic leukaemia (B-CLL) samples showed that, in most cases, fludarabine has an additive cytotoxic activity with rituximab and complement. This report gives a rational support for clinical studies with combinations of drugs, including monoclonal antibodies and fludarabine.