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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Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") can establish the hematopoietic microenvironment within heterotopic ossicles generated by transplantation at non-skeletal sites. Here we show that non-mineralized cartilage pellets formed by hBMSCs ex vivo generate complete ossicles upon heterotopic transplantation in the absence of exogenous scaffolds. These ossicles display a remarkable degree of architectural fidelity, showing that an exogenous conductive scaffold is not an absolute requirement for bone formation by transplanted BMSCs. Marrow cavities within the ossicles include erythroid, myeloid and granulopoietic lineages, clonogenic hematopoietic progenitors and phenotypic HSCs, indicating that complete stem cell niches and hematopoiesis are established. hBMSCs (CD146(+) adventitial reticular cells) are established in the heterotopic chimeric bone marrow through a unique process of endochondral bone marrow formation, distinct from physiological endochondral bone formation. In this process, chondrocytes remain viable and proliferate within the pellet, are released from cartilage, and convert into bone marrow stromal cells. Once explanted in secondary culture, these cells retain phenotype and properties of skeletal stem cells ("MSCs"), including the ability to form secondary cartilage pellets and secondary ossicles upon serial transplantation. Ex vivo, hBMSCs initially induced to form cartilage pellets can be reestablished in adherent culture and can modulate gene expression between cartilage and stromal cell phenotypes. These data show that so-called "cartilage differentiation" of BMSCs in vitro is a reversible phenomenon, which is actually reverted, in vivo, to the effect of generating stromal cells supporting the homing of hematopoietic stem cells and progenitors.

In spite of the remarkable progress in basic and preclinical studies of acute myeloid leukemia (AML), the five-year survival rate of AML patients remains poor, highlighting the urgent need for novel and synergistic therapies. Over the past decade, increased attention has been focused on identifying suitable immunotherapeutic strategies for AML, and in particular on targeting leukemic cells and their progenitors. However, recent studies have also underlined the important contribution of the leukemic microenvironment in facilitating tumor escape mechanisms leading to disease recurrence. Here, we describe the immunological features of the AML niche, with particular attention to the crosstalk between the AML blasts and the cellular components of the altered tumor microenvironment (TME) and the mechanisms of immune escape that hamper the therapeutic effects of the most advanced treatments. Considering the AML complexity, immunotherapy approaches may benefit from a rational combination of complementary strategies aimed at preventing escape mechanisms without increasing toxicity.

Key Points Allogeneic BMT into newborn MPS I mice allows high donor-derived hematopoietic engraftment and prevents bone deformities. Bones of transplanted MPS I mice show significant improvements at radiographic, microcomputed tomography, and histological analyses.

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.

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 g/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 CIKmediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL. (Blood. 2011;117(2):510-518) IntroductionCytokine-induced killer (CIK) cells are activated T cells with natural killer (NK) properties that can be expanded in vitro in presence of recombinant human interleukin-2 (rhIL-2) starting from peripheral blood mononuclear cells stimulated by interferon-␥ and anti-CD3 antibody. 1 They express CD3 and CD56 as well as the NKG2D antigen and show major histocompatibility complex (MHC)-unrestricted cytotoxicity toward neoplastic but not normal targets. 2-5 CIK cells express several chemokine receptors, and in vivo models suggest that they can migrate to the site of tumors after intravenous administration, [6][7][8][9][10] there carrying out their cytotoxic potential and helping to control tumor growth.The ease of CIK cell production in vitro and the cells' antitumor potential have made them suitable candidates for cell therapy programs in solid and hematopoietic tumor treatment. Indeed, both autologous and allogeneic CIK cells have been used in phase I and II clinical trials for the treatment of different tumor types. In these trials, they have shown limited toxicity in vivo, and evidence of antitumor activity has been seen. [11][12][13][14][15][16][17][18] CIK cells have shown cytotoxic activity in vitro and in vivo against hematopoietic neoplastic cells, including AML (acute myeloid leukemia), CML (chronic myelogenous leukemia), and CLL (chronic lymphocytic leukemia). 9,[19][20][21][22] Their cytotoxicity against B-NHL (B-cell nonHodgkin lymphoma), however, has not been fully investigated in vitro, although in vivo murine models suggest they may be active. 1,23 Other biologic treatments available for B-NHL are the anti-CD20 antibodies, for instance, rituximab, and a newgeneration glycoengineered type II...

Background aimsCord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated.MethodsMSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan.ResultsWe were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan.ConclusionsOur results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering.