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)

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 ...

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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 ¼ 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 Â 10 6 to 365 Â 10 6 hMSCs in PL versus a variable number from 2.7 Â 10 6 to 31 Â 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.

Highlights Narsoplimab down-modulates SARS-CoV-2-induced activation of the lectin pathway and endothelial cell damage. Narsoplimab can reduce the thrombotic risk of Covid-19 patients. All patients treated with narsoplimab improved and survived without any drug-related adverse events.

Late occurring lymphomas could be considered an entity distinct from PTLD, occurring within 1 year of transplant, because they show a histological and clinical presentation similar to lymphomas of immunocompetent subjects, are frequently negative for the EBV genome, are invariably clonal, and may rearrange the c-myc oncogene. New therapeutic strategies are required to reduce the mortality rate, and new modalities of long-lasting immunosuppression are called for.

A retroviral vector has been constructed that contains the human CD20 cDNA under the control of the Moloney murine leukemia virus (Mo-MuLV) LTR. Freshly isolated mononuclear cells are infected for three consecutive days in the presence of PHA and hrlL-2, and a mean 15.9% of the cells (range, 6.5 to 31.7%) acquire a CD3+CD20+ phenotype. Transduced T lymphocytes grow and expand in vitro for up to 3 weeks like mock-infected cells and, as observed for the T lymphoblastoid CEM cell line, CD20 expression is maintained for several months with no change in the growth curve of the cells. CD20-expressing CEM and fresh T lymphocytes can be positively immunoselected on columns using different anti-CD20 antibodies. Exposure to monoclonal chimeric anti-CD20 IgG1(kappa) Rituximab antibody (Roche), in the presence of complement, results in effective and rapid killing of the transduced CD3+CD20+ human T cells in vitro. This approach represents a new and alternative method to gene manipulation with "suicide" genes for the production of drug-responsive T cell populations, a crucial step for the future management of graft-versus-host disease in bone marrow transplant patients.