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Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo., (© 2017 by The American Society of Hematology.)

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100., (Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

We have investigated combining adoptive immunotherapy with cytokine-induced killer (CIK) cells and anti-CD20 monoclonal antibodies (mAb) GA101 or rituximab to optimize B-cell non-Hodgkin lymphoma (B-NHL) therapy. CIK cultures alone demonstrated significant cytotoxic activity against B-NHL cell lines or freshly isolated samples in either an autologous or allogeneic combination. This natural cytotoxicity (NC) was mainly due to the predominating CD3(+)CD56(+) CIK population (40%-75%) present in the cultures. The addition of anti-CD20 mAb GA101 or rituximab further increased cytotoxicity by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity (ADCC) mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum (HS) inhibited NK-cell activation induced by rituximab, but not activation induced by GA101.Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 μg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. This was due to the combined action of complement-mediated cytotoxicity (CDC), ADCC, and CIK-mediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL.

Objective: The aim of the work was to determine and characterize, in vitro and in vivo, the therapeutic activity of PHA-793887, a new potent pan-cdk inhibitor, in the context of hematopoietic neoplasms., Materials and Methods: Thirteen leukemic cell lines bearing different cytogenetic abnormalities and normal hematopoietic cells were used in cytotoxicity and colony assays. The drug activity at the molecular level was analyzed by Western blotting. PHA-793887 was also tested in vivo in several leukemia xenograft models., Results: PHA-793887 was cytotoxic for leukemic cell lines in vitro, with IC(50) ranging from 0.3 to 7 microM (mean: 2.9 microM), regardless of any specific chromosomal aberration. At these doses, the drug was not cytotoxic for normal unstimulated peripheral blood mononuclear cells or CD34(+) hematopoietic stem cells. Interestingly, in colony assays PHA-793887 showed very high activity against leukemia cell lines, with an IC(50) <0.1 microM (mean: 0.08 microM), indicating that it has efficient and prolonged antiproliferative activity. PHA-793887 induced cell-cycle arrest, inhibited Rb and nucleophosmin phosphorylation, and modulated cyclin E and cdc6 expression at low doses (0.2-1 microM) and induced apoptosis at the highest dose (5 microM). It was also effective in vivo in both subcutaneous xenograft and primary leukemic disseminated models that better mimic naturally occurring human disease. Interestingly, in one disseminated model derived from a relapsed Philadelphia-positive acute lymphoid leukemia patient, PHA-793887 showed strong therapeutic activity also when treatment was started after establishment of high disease burden., Conclusions: We conclude that PHA-793887 has promising therapeutic activity against acute leukemias in vitro and in vivo.

Background: Chemokines and their receptors are a large family of molecules that control the trafficking of immune cells during their development and in response to inflammation. Non Hodgkin's lymphoma (NHL) derives from the neoplastic transformation of lymphocytes at different stages of differentiation and may show systemic, nodal and extranodal localisation as well as metastasis in different sites., Objective: To investigate the role of the chemokine system in the pathology of NHL and as a potential drug target in this disease., Method: The expression of chemokines and receptors by different NHL subtypes as well as their likely functional role in terms of lymphoma tissue localisation, lymphoma growth, tumour angiogenesis and recruitment of immune cells are reviewed. The data regarding antagonists or chemokines as potential therapeutic agents for NHL is discussed., Results/conclusions: NHL's express functional chemokine receptors, which, at least in part, dictate tissue localisation and perhaps metastatic potential. Different types of chemokine antagonists have been tested in vitro and in vivo and several peptide or small molecule inhibitors have reached Phase I/II clinical trials for different diseases. Although the field is in its early days, interesting compounds are being developed, directed especially against homeostatic chemokine receptors.

Background and Objectives: The therapeutic antibody alemtuzumab is directed against the CD52 molecule and is used for the treatment of B-cell lymphocytic leukemia (B-CLL). We investigated the mechanism of action of this antibody in vitro against different neoplastic B cells and compared it to the anti-CD20 antibody rituximab., Design and Methods: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells using human serum as the source of complement. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated by chromium release assays, using peripheral blood mononuclear cells as effector cells, before and after 2 days of culture with interleukin-2 (IL-2)., Results: Alemtuzumab lysed cells from the 23 B-CLL samples through complement activation (mean 80%) much more efficiently than rituximab did (mean 16%), presumably because of the higher expression of CD52 than of CD20. All other leukemic B cells, including 1 prolymphocytic leukemia, 2 hairy cell leukemias and 6 B-non Hodgkin's lymphomas were effective targets for both antibodies, with 88% and 85% mean lysis, respectively. Both CD52 and CD20 were highly expressed in these cells. In contrast, most neoplastic B cell samples were poorly lysed through ADCC using freshly isolated peripheral blood mononuclear cells as effectors with either monoclonal antibody and regardless of target antigen levels. ADCC was, however, significantly increased in all cases by culturing the effector cells with IL-2 for 2 days., Interpretation and Conclusions: Complement-mediated lysis is likely to be an important mechanism of action of alemtuzumab in B-CLL and combination with IL-2 may increase this antibody's efficacy through ADCC. Mature neoplastic B cells other than B-CLL express high levels of CD52 and are good targets for alemtuzumab-mediated cytotoxicity.

Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20(+) lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20(+) cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa(-/-)). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo.

Rituximab (Mabthera) and alemtuzumab (Campath(R), Mabcampath(R)) are non-conjugated IgG1 therapeutic monoclonal antibodies directed against the CD20 and CD52 surface antigens respectively. They are presently used in the therapy of indolent B-cell non-Hodgkin's lymphoma (B-NHL) and of B-cell chronic lymphocytic leukaemia, and are thought to act mainly through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Here we have analysed the capacity of these two monoclonal antibodies to lyse cell lines of acquired immunodeficiency syndrome (AIDS)-related B-NHL through either complement activation or antibody-dependent cytotoxicity. Rituximab strongly activated both CDC and ADCC against CD20-positive AIDS-NHL cells lines, inducing up to 60-98% and 20% specific lysis respectively. In contrast, alemtuzumab was a poor activator of CDC, even in the AIDS-NHL cell lines expressing high amounts of CD52, leading to a lysis of only 1-30%, whereas it was at least as strong as rituximab in inducing ADCC of the same lines (up to 30% specific lysis). Altogether, these data offer a first in vitro rationale supporting the therapeutic use of rituximab for CD20-positive AIDS-NHL.

Rituximab, the humanized chimeric anti-CD20 monoclonal antibody, represents a powerful tool for treating B-cell malignancies and is licensed for the treatment of relapsed or chemorefractory low-grade or follicular non-Hodgkin's lymphoma (NHL). It has a unique mode of action and can induce killing of CD20+ cells via multiple mechanisms. The direct effects of rituximab include complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and the indirect effects include structural changes, apoptosis, and sensitization of cancer cells to chemotherapy. In vitro studies have made a significant contribution to the understanding of these mechanisms of action and have led to the development of innovative and effective treatment strategies to optimize patient response. The most significant of these strategies is the combination of rituximab and CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine and prednisone), which is proving a highly effective combination in the treatment of NHL. However, all patients do not respond equally well to rituximab, and in vitro studies have identified a possible mechanism of resistance involving the anti-complement inhibitors CD55 and CD59. Neutralizing antibodies to CD55 and CD59 can overcome resistance to rituximab-mediated complement-mediated cytotoxicity in vitro. This paper overviews our understanding of the mechanisms of action of rituximab and identifies how this knowledge could be applied in a clinical setting to maximize response in both sensitive and resistant patients.

The therapeutic unconjugated anti-CD20 Mab rituximab is used for the treatment of B-non-Hodgkin’s lymphomas. We have studied the direct biological effects, signalling and gene expression profiles induced by rituximab in two human B-lymphoma cell lines, DHL4 and BJAB, using microarray, quantitative PCR and gel shift analysis. Rituximab alone inhibited thymidine uptake and induced homotypic adhesion in DHL4 only, but not BJAB. Analysis of Affymetrix microchips carrying probes for about 10,000 human cDNAs, allowed us to identify 16 genes in DHL4 and 12 in BJAB induced by rituximab at 4 h. Eleven and seven of these genes were specific for DHL4 and BJAB, respectively; whereas the remaining five were up-regulated in both cell lines. Mean induction ranged from 2- to 16-fold. Real time PCR analysis allowed us to confirm up-regulation of all genes identified, except one in BJAB. Time course of induction of eight genes was studied, showing peak induction in most cases at 4 h. The up-regulation of 5/5 genes was also observed with the F(ab')2 fragment of rituximab. Analysis of three further B-cell lymphoma lines showed that gene induction is not restricted to BJAB and DHL4. Finally, we show that rituximab alone can induce AP1 activation in both cell lines and provide evidence that the ERK1/2 pathway is involved in the rituximab-mediated up-regulation of gene expression. These data demontrate that rituximab alone has direct signalling capacity in different B-lymphoma lines, inducing distinct but overlapping sets of genes which may play a role in the biological and/or therapeutic effect of the antibody. [ABSTRACT FROM AUTHOR]