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. (Blood. 2001;98:3383-3389)

SummaryBackground and objectives Mesenchymal stromal cells (MSCs) abrogate alloimmune response in vitro, suggesting a novel cell-based approach in transplantation. Moving this concept toward clinical application in organ transplantation should be critically assessed.Design, setting, participants & measurements A safety and clinical feasibility study (ClinicalTrials.gov, NCT00752479) of autologous MSC infusion was conducted in two recipients of kidneys from living-related donors. Patients were given T cell-depleting induction therapy and maintenance immunosuppression with cyclosporine and mycophenolate mofetil. On day 7 posttransplant, MSCs were administered intravenously. Clinical and immunomonitoring of MSC-treated patients was performed up to day 360 postsurgery.Results Serum creatinine levels increased 7 to 14 days after cell infusion in both MSC-treated patients. A graft biopsy in patient 2 excluded acute graft rejection, but showed a focal inflammatory infiltrate, mostly granulocytes. In patient 1 protocol biopsy at 1-year posttransplant showed a normal graft. Both MSCtreated patients are in good health with stable graft function. A progressive increase of the percentage of CD4 ϩ CD25 high FoxP3 ϩ CD127 Ϫ Treg and a marked inhibition of memory CD45RO ϩ RA Ϫ CD8 ϩ T cell expansion were observed posttransplant. Patient T cells showed a profound reduction of CD8 ϩ T cell activity. ConclusionsFindings from this study in the two patients show that MSC infusion in kidney transplant recipients is feasible, allows enlargement of Treg in the peripheral blood, and controls memory CD8 ϩ T cell function. Future clinical trials with MSCs to look with the greatest care for unwanted side effects is advised.

ABSTRACT

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

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Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcγRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-γ/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-γ had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.

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IntroductionCytokine-induced killer (CIK) cells are ex vivo-activated lymphocytes that can be obtained in large numbers within 3 weeks of culture from either human peripheral blood or BM, or cord blood mononuclear cells by the sequential addition of IFN-␥, anti-CD3 Ab (OKT3), and high doses of recombinant human IL-2 (rhIL-2). 1-4 CIK cells represent an heterogeneous cell population, including a large majority of CD3 ϩ CD56 ϩ cells and minor fractions of typical T cells (CD3 ϩ CD56 Ϫ ) and NK cells (CD3 Ϫ CD56 ϩ ). 5,6 CIK cells can lyse a broad array of tumor targets, including acute myeloid leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia by a non-MHC-restricted, natural killer (NK)-like mechanism. 3,6 Interestingly, in mice, CIK cells display negligible alloreactivity and cause minimal GVHD compared with allogeneic splenocytes, when infused after allogeneic BM transplantation in murine models. 7,8 CIK cells express several chemokine receptors and can migrate to the site of tumors after intravenous administration, as shown by models in vivo. [9][10][11][12][13] Because CIK cells could be produced by a simple approach and displayed antitumor activity in vitro, they appeared to be suitable candidates for cell therapy in solid and hematopoietic tumor treatment. Indeed, both autologous and allogeneic CIK cells have been used in phase 1 and 2 clinical trials for the treatment of different tumor types. In these trials, they displayed limited toxicity, whereas evidence has been obtained that they exert antitumor activity. [14][15][16][17][18][19][20] The molecular structures that account for tumor recognition and killing by CIK cells are only partially understood. Previous observations suggested a possible involvement of the NKG2D and lymphocyte function-associated antigen-1 (LFA-1) molecules [21][22][23] ; however, little is known on the role of other activating NK receptors, including DNAX accessory molecule-1 (DNAM-1), NKp30, NKp44, NKp46, as well as on the involvement of TCR/CD3 complex in the cytolytic activity of CIK cells.Our previous studies clarified that CD3 ϩ CD56 ϩ CIK cells have phenotypic characteristics typical of terminally differentiated CD8 ϩ effector memory T cells (T EMRA ; CCR7 Ϫ , CD45RA ϩ , CD62L low , CD11a ϩ , CD27 ϩ , CD28 Ϫ ). We also showed that CIK cells originate in vitro from CD56 Ϫ CD8 ϩ T-cell progenitors that strongly expand on culture in the presence of IL-2 and acquire CD56 antigen. 5 In addition, CIK cells share several characteristics with NK cells, such as the large granular lymphocyte structure, the capacity to kill the HLA class I-negative cell line K562, and the surface expression of high densities of CD56 and NKG2D. Different from NK cells, however, CIK cells express low densities of NKp30, whereas they do not express NKp44 and NKp46, and the inhibitory killer immunoglobulin-like receptors, NKG2A and CD94. 5 In the present study, we investigated in detail the receptors involved in the NK-like cytolytic activity of CIK cells and analyzed whether they ...